Method of treating cancer by administering multivalent fibronectin based scaffold domain proteins

ABSTRACT

The present invention relates to multivalent polypeptides comprising at least two fibronectin scaffold domains connected via a polypeptide linker. The invention also relates to multivalent polypeptides for use in diagnostic, research and therapeutic applications. The invention further relates to cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the innovative proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/229,415, filed Mar. 28, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/533,382, filed Jun. 26, 2012 (now U.S. Pat. No.8,728,483), which is a continuation of U.S. patent application Ser. No.12/470,989, filed May 22, 2009 (now U.S. Pat. No. 8,221,765), whichclaims the benefit of U.S. Provisional Application Nos. 61/128,651,filed May 22, 2008; 61/212,982, filed Apr. 17, 2009; and 61/178,395,filed May 14, 2009. The contents of the aforementioned applications arehereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 10, 2018, isnamed MXI_521CN2CN_Sequence_Listing.txt and is 64,581 bytes in size.

FIELD OF THE INVENTION

The present invention relates to multivalent fibronectin based scaffolddomains for use in diagnostic, research and therapeutic applications.The invention further relates to cells comprising such proteins,polynucleotide encoding such proteins or fragments thereof, and tovectors comprising the polynucleotides encoding the innovative proteins.

INTRODUCTION

Fibronectin based scaffolds are a family of proteins capable of evolvingto bind any compound of interest. These proteins, which generally makeuse of a scaffold derived from a fibronectin type III (Fn3) or Fn3-likedomain, function in a manner characteristic of natural or engineeredantibodies (that is, polyclonal, monoclonal, or single-chain antibodies)and, in addition, possess structural advantages. Specifically, thestructure of these antibody mimics has been designed for optimalfolding, stability, and solubility, even under conditions that normallylead to the loss of structure and function in antibodies. An example offibronectin-based scaffold proteins are Adnectins™ (Adnexus, aBristol-Myers Squibb R&D Company).

Fibronectin is a large protein which plays essential roles in theformation of extracellular matrix and cell-cell interactions; itconsists of many repeats of three types (types I, II, and III) of smalldomains (Baron et al., 1991). Fn3 itself is the paradigm of a largesubfamily which includes portions of cell adhesion molecules, cellsurface hormone and cytokine receptors, chaperoning, andcarbohydrate-binding domains. For reviews see Bork & Doolittle, ProcNatl Acad Sci USA. 1992 Oct. 1; 89(19):8990-4; Bork et al., J Mol Biol.1994 Sep. 30; 242(4):309-20; Campbell & Spitzfaden, Structure. 1994 May15; 2(5):333-7; Harpez & Chothia, J Mol Biol. 1994 May 13;238(4):528-39).

Fibronectin type III (Fn3) domains comprise, in order from N-terminus toC-terminus, a beta or beta-like strand, A; a loop, AB; a beta orbeta-like strand, B; a loop, BC; a beta or beta-like strand, C; a loop,CD; a beta or beta-like strand, D; a loop, DE; a beta or beta-likestrand, E; a loop, EF; a beta or beta-like strand, F; a loop, FG; and abeta or beta-like strand, G. Any or all of loops AB, BC, CD, DE, EF andFG may participate in target binding. The BC, DE, and FG loops are bothstructurally and functionally analogous to the complementaritydetermining regions (CDRs) from immunoglobulins.

U.S. Pat. No. 7,115,396 describes Fn3 domain proteins whereinalterations to the BC, DE, and FG loops result in high affinity TNFαbinders. U.S. Publication No. 2007/0148126 describes Fn3 domain proteinswherein alterations to the BC, DE, and FG loops result in high affinityVEGFR2 binders.

It would be advantageous to obtain improved fibronectin domain scaffoldproteins for both therapeutic and diagnostic purposes. The presentdisclosure provides such improved proteins.

SUMMARY OF THE INVENTION

One aspect of the application provides for multivalent polypeptidescomprising an N-terminal domain comprising a first fibronectin type IIItenth domain (¹⁰Fn3) that binds to a first target molecule with a K_(D)of less than 500 nM and a C-terminal domain comprising a second ¹⁰Fn3that binds to a second target molecule with a K_(D) of less than 500 nM.In some embodiments, first and second ¹⁰Fn3 domains are linked via apolypeptide linker, such as, for example, a polypeptide having from1-100, 1-50, 1-20, 1-10, 5-50, 5-20, 5-10, 10-50, or 10-20 amino acids.In some embodiments, first and second ¹⁰Fn3 domains are linked via apolypeptide selected from a glycine-serine based linker, such as SEQ IDNOS: 21 and 22. In some embodiments, the first and second ¹⁰Fn3 domainsare linked via the polypeptide depicted in SEQ ID NO: 20. In someembodiments, first and second ¹⁰Fn3 domains are linked via a polypeptideselected from a glycine-proline based linker, such as SEQ ID NOS: 32,33, and 34. In some embodiments, first and second ¹⁰Fn3 domains arelinked via a polypeptide selected from a proline-alanine based linker,such as SEQ ID NOS: 60, 61 and 62.

The ¹⁰Fn3 domains each comprise a loop, AB; a loop, BC; a loop, CD; aloop, DE; a loop, EF; and a loop, FG and each, independently, have atleast one loop selected from loop BC, DE, and FG with an altered aminoacid sequence relative to the sequence of the corresponding loop of thehuman ¹⁰Fn3 domain. The ¹⁰Fn3 domains each comprise an amino acidsequence that is at least 50, 60, 70, or 80% identical to the naturallyoccurring human ¹⁰Fn3 domain represented by SEQ ID NO: 1.

In some embodiments, the first ¹⁰Fn3 domain binds to a first targetmolecule with a K_(D) of less than 100 nM and the second ¹⁰Fn3 domainbinds to a second target molecule with a K_(D) of less than 100 nM. Insome embodiments, the first target molecule and the second targetmolecule are the same. In some embodiments, the first target moleculeand the second target molecule are different. In some embodiments, thefirst target molecule is IGF-IR and the second target molecule isVEGFR2. In some embodiments, the first target molecule is VEGFR2 and thesecond target molecule is IGF-IR.

In some embodiments, at least two loops of the ¹⁰Fn3 domains arealtered. In some embodiments, loop BC and loop FG have an altered aminoacid sequence relative to the sequence of the corresponding loop of thehuman ¹⁰Fn3 domain. In some embodiments, at least three loops of the¹⁰Fn3 domains are altered. In some embodiments, at least two loops ofthe ¹⁰Fn3 domain bind a target molecule. In some embodiments, threeloops of the ¹⁰Fn3 domain bind a target molecule.

In some embodiments, the first and/or second ¹⁰Fn3 domain is linked atits C-terminus to an amino acid sequence selected from: SEQ ID NO: 17,18, 19, 50, 51, 52, 71 or 72 or E, EI, EID, ES, EC, EGS, or EGC. In someembodiments, the first ¹⁰Fn3 domain is linked at its C-terminus to theamino acid sequence of SEQ ID NO: 19 or 50, or E, EI, or EID. In someembodiments, the second ¹⁰Fn3 domain is linked at its C-terminus to theamino acid sequence of SEQ ID NO: 17, 18, 51, 52, 71 or 72.

In some embodiments, multivalent polypeptides are provided comprisingthe amino acid sequence at least 70, 80, 90, 95, or 100% identical toany one of SEQ ID NOS: 8-15, 29-31 and 63-70.

In some embodiments, the multivalent polypeptide further comprises oneor more pharmacokinetic (PK) moieties selected from: a polyoxyalkylenemoiety, a human serum albumin binding protein, sialic acid, human serumalbumin, transferrin, IgG, an IgG binding protein, and an Fc fragment.In some embodiments, the PK moiety is the polyoxyalkylene moiety andsaid polyoxyalkylene moiety is polyethylene glycol (PEG). In someembodiments, the PEG moiety is covalently linked to the multivalentpolypeptide via a Cys or Lys amino acid. In some embodiments, the PEG isbetween about 0.5 kDa and about 100 kDa.

In some embodiments, the PK moiety improves one or more pharmacokineticproperties of the polypeptides, e.g., bioavailability, serum half-life,in vivo stability, and drug distribution. In some embodiments, the PKmoiety increases the serum half-life of the multivalent polypeptide byat least 20, 30, 40, 50, 70, 90, 100, 120, 150, 200, 400, 600, 800% ormore relative to the multivalent polypeptide alone. In some embodiments,the multivalent polypeptide further comprising a PK moiety has a serumin vivo half-life of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,or 14 days.

In some embodiments, the PK moiety and the ¹⁰Fn3 domain are linked viaat least one disulfide bond, a peptide bond, a polypeptide, a polymericsugar, or a polyethylene glycol moiety. In some embodiments, the PKmoiety and the ¹⁰Fn3 domain are linked via a polypeptide comprising theamino acid sequence of SEQ ID NOS: 17, 18, or 20.

In some embodiments, the multivalent polypeptide further comprises abinding moiety. In some embodiments, the multivalent polypeptide furthercomprises an antibody moiety. In some embodiments, the antibody moietyis less than 50 KDa. In some embodiments, the antibody moiety is lessthan 40 KDa. In some embodiments, the antibody moiety is a single chainFvs (scFvs), Fab fragment, Fab′ fragment, F(ab′)2, disulfide linked Fv(sdFv), Fv, diabody, or whole antibody. In some embodiments, theantibody moiety is a single domain antibody. In some embodiments, theantibody moiety binds a human protein. In some embodiments the antibodymoiety binds IGF-IR, FGFR1, FGFR2, FGFR3, FGFR4, c-Kit, human p185receptor-like tyrosine kinase, HER2, HER3, c-Met, folate receptor,PDGFR, VEGFR1, VEGFR2, VEGFR3, human vascular endothelial growth factor(VEGF) A, VEGF C, VEGF D, human CD20, human CD18, human CD11a, humanapoptosis receptor-2 (Apo-2), human alpha4beta7 integrin, humanGPIIb-IIIa integrin, stem cell factor (SCF), EGFR, or human CD3.

In some embodiments, the multivalent polypeptide further comprises aderivative of lipocalin; a derivative of tetranectin; an avimer; or aderivative of ankyrin. In some embodiments, the multivalent polypeptidebinds a human protein. In some embodiments the multivalent polypeptidebinds IGF-IR, FGFR1, FGFR2, FGFR3, FGFR4, c-Kit, human p185receptor-like tyrosine kinase, HER2, HER3, c-Met, folate receptor,PDGFR, VEGFR1, VEGFR2, VEGFR3, human vascular endothelial growth factor(VEGF) A, VEGF C, VEGF D, human CD20, human CD18, human CD11a, humanapoptosis receptor-2 (Apo-2), human alpha4beta7 integrin, humanGPIIb-IIIa integrin, stem cell factor (SCF), EGFR, or human CD3.

In some embodiments, the multivalent polypeptide further comprises abinding moiety linked via at least one disulfide bond, a peptide bond, apolypeptide, a polymeric sugar, or a polyethylene glycol moiety (PEG).In some embodiments, the PEG is between about 0.5 kDa and about 100 kDa.In some embodiments, the PEG is conjugated to the polypeptide and thebinding moiety via a Cys or Lys residue.

In one aspect, the application provides a multivalent polypeptide thathas been deimmunized to remove one or more T-cell epitopes. In oneaspect, the application provides a multivalent polypeptide that has beendeimmunized to remove one or more B-cell epitopes.

In one aspect, the application provides a multivalent polypeptidewherein the ¹⁰Fn3 domains are selected by the method comprising a)producing a population of candidate nucleic acid molecules, eachcomprising a candidate fibronectin type III (¹⁰Fn3) domain sequencewhich differs from human ¹⁰Fn3 domain coding sequence, said nucleic acidmolecules each comprising a translation initiation sequence and a startcodon operably linked to said candidate ¹⁰Fn3 domain coding sequence andeach being operably linked to a nucleic acid-puromycin linker at the 3′end; b) in vitro translating said candidate ¹⁰Fn3 domain codingsequences to produce a population of candidate nucleic acid-¹⁰Fn3fusions; c) contacting said population of candidate nucleic acid-¹⁰Fn3fusions with a target molecule; and d) selecting a nucleic acid-¹⁰Fn3fusion, the protein portion of which has a binding affinity orspecificity for the target molecule that is altered relative to thebinding affinity or specificity of said human ¹⁰Fn3 for the targetmolecule. In some embodiments, the selected nucleic acid-¹⁰Fn3 fusion isfurther optimized by altering one or more nucleic acid residues andrescreening the fusion with the target molecule to select for improvedbinders. In some embodiments the candidate nucleic acid molecule is RNA.In some embodiments the candidate nucleic acid molecule is DNA.

In one aspect, the application provides pharmaceutically acceptablecompositions comprising a multivalent polypeptide. In some embodiments,the composition is essentially endotoxin free. In some embodiments, thecomposition is substantially free of microbial contamination making itsuitable for in vivo administration. The composition may be formulated,for example, for IV, IP or subcutaneous administration.

In another aspect, the application provides a nucleic acid encoding amultivalent polypeptide as described herein. Vectors containingpolynucleotides for such proteins are included as well. Suitable vectorsinclude, for example, expression vectors. A further aspect of theapplication provides for a cell, comprising a polynucleotide, vector, orexpression vector, encoding a multivalent polypeptide. Sequences arepreferably optimized to maximize expression in the cell type used.Preferably, expression is in E. coli. Multivalent polypeptides can alsobe expressed, for example, in eukaryotic microbes, including yeast(e.g., pichia or cerevisiae) or blue green algae. Yeast cells can beengineered to produce desired glycosylations. The cells of the inventioncan be a mammalian cell. In one aspect, the mammalian cell can beengineered to produce desired glycosylations. In one aspect, the cellexpresses a fibronectin based scaffold protein. In one aspect, thepolynucleotides encoding fibronectin based scaffold proteins are codonoptimized for expression in the selected cell type. Also provided aremethods for producing a multivalent polypeptide as described herein,comprising culturing a host cell comprising a nucleic, vector, orexpression vector, comprising a nucleic acid encoding the multivalentpolypeptide and recovering the expressed multivalent polypeptide fromthe culture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Various HTPP purified V/I ¹⁰Fn3-based binders demonstratedsimilar expression profiles. As shown: ‘GS5’ (SEQ ID NO: 21) and ‘GS10’(SEQ ID NO: 22).

FIG. 2. SEC of HTPP purified construct 385A08-Fn-V2B demonstratespredominantly monomeric protein

FIG. 3. SEC of HTPP purified construct V2B-Fn-385A08 demonstrates amixture of monomeric and dimeric protein

FIG. 4. Molecular Weight as measured by LC-MS and Tm determination byDSC for certain V/I ¹⁰Fn3-based binders. As shown: ‘GS10’ (SEQ ID NO:22).

FIG. 5. SEC of 385A08-Fn-V2B-cys (pegylated, no his-tag) demonstratesthat this protein is 95.1% monomeric.

FIG. 6. DSC analysis of 385A08-Fn-V2B-cys (pegylated, no his-tag)demonstrated that the protein was half unfolded at 56° C., and nounfolding was detected up to 45° C.

FIG. 7. Mice dosed with a 40 kD branched or linear pegylated version ofconstruct 385A08-Fn-V2B-Cys demonstrated half life of 14.6 hours forbranched vs. 10.5 hours for linear. Several individual mice were dosedwith the branched or linear pegylated version of the construct asindicated in the figure.

FIG. 8. VEGFR2/IGF-IR tandems are equipotent in cells to VEGFR2 orIGF-IR individual binders. Constructs were tested in Biacore assay forbinding to IGF-IR, and in Ba/F3 and Rh41 assays to determine IC50 (seeExamples 5 and 7). The sequences of the molecular forms are described inExample 1. As shown: ‘GS5’ (SEQ ID NO: 21) and ‘GS10’ (SEQ ID NO: 22).

FIG. 9. Certain V/I ¹⁰Fn3-based binders were evaluated for their kineticbehavior towards IGF-IR and VEGFR2. As shown: ‘GS10’ (SEQ ID NO: 22).

FIG. 10. His-tag and non-his-tag versions of the pegylated construct385A08-Fn-V2B-cys were evaluated for their kinetic behavior towardsIGF-IR and VEGFR2.

FIG. 11. The relative ability of Peg-V2BShort and 385A08-Fn-V2B-cys(with PEG and his-tag) to compete with labeled Peg-V2BShort for cellbound VEGFR-2 was evaluated on the cell line 293:KDR

FIG. 12. The relative ability of AT580-PEG40 and 385A08-Fn-V2B-cys (withPEG and his-tag) to compete with labeled AT580-PEG20-AT580 for cellbound IGF-IR was evaluated on the cell line R+.

FIG. 13. V/I ¹⁰Fn3-based binders were evaluated in the cell based assaysRh41 and HMVEC-L. As shown: ‘GS5’ (SEQ ID NO: 21) and ‘GS10’ (SEQ ID NO:22).

FIG. 14. Selected constructs, including his-tag and non-his-tag of thepegylated construct 385A98-Fn-V2B-Cys were evaluated for their activityin the cell based assays NCI-H929 and Ba/F3, compared to controls.

FIG. 15. HEK293/KDR and PAE/KDR cells express VEGFR2 and IGF-IR and bothreceptors are activated in the presence of their cognate ligands. HEK293and PAE cells transfected with KDR (VEGFR2) were stimulated with VEGF,IGF1, or both factors (VEGF/IGF1). Western blots of the cell lysateswere probed for phosphorylated VEGFR (p-Flk-1/Tyr996), phosphorylatedIGF-IR, phosphorylated Akt, phosphorylated MAPK, or total actin.

FIG. 16. Fibronectin based scaffold multimers block VEGFR2 and IGF-IRligand induced phosphorylation and inhibit proliferation of HEK293/KDRcells. Cells were treated with VEGF and IGF1 (“−” indicatesnon-stimulated control) and fibronectin scaffold domain proteins asdescribed in Example 8. Western blots of cell lysates were probed forphosphorylated VEGFR (p-Flk-1/Tyr996), total VEGFR (Flk-1),phosphorylated IGF-IR, total IGF-IR, phosphorylated Akt, total Akt,phosphorylated MAPK, total MAPK, or total actin. Cell proliferation wasevaluated by [³H]-thymidine incorporation after exposure to the variousconstructs and the results reported as IC₅₀. As shown: ‘GS5’ (SEQ ID NO:21) and ‘GS10’ (SEQ ID NO: 22).

FIG. 17. Fibronectin based scaffold multimers block VEGFR2 and IGF-IRligand induced phosphorylation and inhibit proliferation of HEK293/KDRcells. Cells were treated with VEGF and IGF1 and fibronectin scaffolddomain proteins as described in Example 8. Western blots of cell lysateswere probed for phosphorylated VEGFR (p-Flk-1/Tyr996), total VEGFR(Flk-1), phosphorylated IGF-IR, total IGF-IR, phosphorylated Akt, totalAkt, phosphorylated MAPK, total MAPK, or total actin. Cell proliferationwas evaluated by [³H]-thymidine incorporation after exposure to thevarious constructs and the results reported as IC₅₀. As shown: ‘GS5’(SEQ ID NO: 21) and ‘GS10’ (SEQ ID NO: 22).

FIG. 18. Cellular Proliferation Assay: Effect of various V/I ¹⁰Fn3-basedbinders in HMVEC cells after exposure for 72 hrs.

FIG. 19. Western blot analysis: HMVEC-L cells were exposed to increasingconcentrations of various constructs for 1 hr followed by activationwith VEGF/IGF-1 ligands (both 50 ng/ml final concentration) for 10 minat 37° C. prior to lysis. Inhibition of pIGF-1R, pAkt, and pMAPKactivities were measured by comparing the ratio of phospho-signals inuntreated versus treated samples, and after normalization to GAPDH whichis used as a loading control.

FIG. 20. Western blot analysis: Rh41 cells were exposed to increasingconcentrations of compounds for 1 hr followed by activation with IGF-1ligand (50 ng/ml) for 10 min at 37° C. prior to lyses. pIGF-1R and pAktactivities were measured by comparing the ratio of phospho-signals inuntreated versus treated samples, and after normalization to GAPDH whichis used as a loading control.

FIG. 21. Effect of various constructs on calcium release in HMVEC-Lcells after exposure for 1 hr. Cells were serum-starved overnight in EBMmedia and then incubated in the presence of increasing amounts ofvarious constructs for 1 hour. The cells were stimulated with 50 ng/mlVEGF ligand. Ca²⁺ flux was measured 60 seconds after ligand addition.

FIGS. 22A and 22B. Antitumor activity of pegylated his-tagged385A08-Fn-V2B-cys in the RH-41 tumor xenograft model at a single doselevel. FIG. 22A. Growth curve following a 3×wk×3 regimen. FIG. 22B.Percent growth inhibition plot demonstrating greater than 50% TGI overone TVDT when compared to vehicle treated group. (▾) Indicates dosingregimen.

FIGS. 23A-23D. Antitumor activity of pegylated, his-tagged385A08-Fn-V2B-cys in the RH-41 tumor xenograft model at multiple doselevels. FIGS. 23A-23D list the indicated dose levels of each agent. (▾)Indicates dosing regimen.

FIGS. 24A-24D. Antitumor activity of pegylated, his-tagged385A08-Fn-V2B-cys in the A549 tumor xenograft lung model at multipledose levels. The indicated dose levels are shown in FIGS. 24A and 24Cwith corresponding % TGI in FIGS. 24B and 24D. (▾) Indicates dosingregimen.

FIGS. 25A-25D. Antitumor activity of pegylated, his-tagged385A08-Fn-V2B-cys in the GEO tumor xenograft colon model at multipledose levels. The indicated dose levels are shown in FIGS. 25A and 25Cwith corresponding % TGI in FIGS. 25B and 25D. (▾) Indicates dosingregimen.

FIG. 26. pegylated, his-tagged 385A08-Fn-V2B-cys mediated Tumor GrowthInhibition in A673 Ewing Sarcoma Xenograft Tumors is comparable to thatof the mononectin PEG-V2B-short, a pegylated anti-VEGFR-2 adnectin.

FIG. 27. Pharmacokinetic parameters of 385A08-Fn-V2B-cys (with his).

FIGS. 28A-28E. Fitted vs. observed plasma concentration-time profiles of385A08-Fn-V2B-cys (with his) and mVEGF-A after IP administration of 20and 200 mg/kg to nude mice bearing the A673 tumor.

FIGS. 29A-29E. Fitted vs. observed plasma concentration-time profiles of385A08-Fn-V2B-cys (no his) and mVEGF-A after IP administration of 20 and200 mg/kg to nude mice bearing the A673 tumor.

FIG. 30. Pharmacokinetic parameters of 385A08-Fn-V2B-cys (with his) inmonkeys.

FIGS. 31A-31H. Fitted vs. observed plasma concentration-time profiles of385A08-Fn-V2B-cys (with his) and hVEGF-A after IV administration of 3and 30 mg/kg to monkeys.

FIG. 32. Pharmacokinetic parameters of 385A08-Fn-V2B-cys (non-histagged) in monkeys.

FIGS. 33A-33F. Fitted vs. observed plasma concentration-time profiles of385A08-Fn-V2B-cys (no his) and hVEGF-A after IV administration of 3mg/kg to monkeys (1st Dose).

DETAILED DESCRIPTION OF THE INVENTION Definitions

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

The term “PK” is an acronym for “pharmokinetic” and encompassesproperties of a compound including, by way of example, absorbtion,distribution, metabolism, and elimination by a subject. A “PK modulationprotein” or “PK moiety” refers to any protein, peptide, or moiety thataffects the pharmokinetic properties of a biologically active moleculewhen fused to or administered together with the biologically activemolecule. Examples of a PK modulation protein or PK moiety include PEG,human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos.20050287153 and 20070003549), human serum albumin, Fc or Fc fragments,and sugars (e.g., sialic acid).

A “functional Fc region” possesses at least one “effector function” of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g., an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g., from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

Targets may also be fragments of said targets. Thus a target is also afragment of said target, capable of eliciting an immune response. Atarget is also a fragment of said target, capable of binding to a singledomain antibody raised against the full length target.

A fragment as used herein refers to less than 100% of the sequence(e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), butcomprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25 or more amino acids. A fragment is of sufficientlength such that the interaction of interest is maintained with affinityof 1×10⁻⁶ M or better.

A fragment as used herein also refers to optional insertions, deletionsand substitutions of one or more amino acids which do not substantiallyalter the ability of the target to bind to a single domain antibodyraised against the wild-type target. The number of amino acid insertionsdeletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thedisorder. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the time todisease progression (TTP) and/or determining the response rates (RR).

The half-life of an amino acid sequence or compound can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thesequence or compound and/or clearance or sequestration of the sequenceor compound by natural mechanisms. The half-life can be determined inany manner known per se, such as by pharmacokinetic analysis. Suitabletechniques will be clear to the person skilled in the art, and may forexample generally involve the steps of suitably administering to theprimate a suitable dose of the amino acid sequence or compound to betreated; collecting blood samples or other samples from said primate atregular intervals; determining the level or concentration of the aminoacid sequence or compound of the invention in said blood sample; andcalculating, from (a plot of) the data thus obtained, the time until thelevel or concentration of the amino acid sequence or compound of theinvention has been reduced by 50% compared to the initial level upondosing. Reference is for example made to to the standard handbooks, suchas Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Peters et al, Pharmacokinete analysis: APractical Approach (1996). Reference is also made to “Pharmacokinetics”,M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition(1982).

Half-life can be expressed using parameters such as the t½-alpha,t½-beta and the area under the curve (AUC). In the presentspecification, an “increase in half-life” refers to an increase in anyone of these parameters, such as any two of these parameters, oressentially all three these parameters. An “increase in half-life” inparticular refers to an increase in the t½-beta, either with or withoutan increase in the t½-alpha and/or the AUC or both.

As used herein, the term “multivalent” refers to a recombinant moleculethat incorporates two or more biologically active segments. The proteinfragments forming the multivalent molecule optionally may be linkedthrough a polypeptide linker which attaches the constituent parts andpermits each to function independently.

Overview

The application provides multivalent polypeptides comprising two or morefibronectin based scaffold proteins, such as Fn3 domains. The Fn3domains may bind to the same target, thereby increasing the valency andthus the avidity of target binding, or to different targets, therebydemonstrating multiple effector functions.

U.S. Pat. No. 6,818,418 describes fibronectin scaffold multimers thatmay be linked covalently or non-covalently. The present applicationdescribes improved multimers that are covalently bonded via polypeptidelinkers, allowing a multivalent polypeptide to be expressed as a singleconstruct. The application relates, in part, on the surprising discoverythat Fn3 domains joined via a polypeptide linker correctly foldindependently of each other, retain high affinity binding, and that eachof the domains retains its functional properties.

Fibronectin Based Scaffolds

One aspect of the application provides for polypeptides comprising atleast two Fn3 domains, each binding a target molecule, linked via apolypeptide. Each Fn3 domain comprises an AB loop, a BC loop, a CD loop,a DE loop, an EF loop, and an FG loop.

In some embodiments, the Fn3 domain is an Fn3 domain derived from humanfibronectin, particularly the tenth Fn3 domain of fibronectin (¹⁰Fn3),as shown in SEQ ID NO: 1. A variety of mutant ¹⁰Fn3 scaffolds have beenreported. In one aspect, one or more of Asp 7, Glu 9, and Asp 23 isreplaced by another amino acid, such as, for example, a non-negativelycharged amino acid residue (e.g., Asn, Lys, etc.). These mutations havebeen reported to have the effect of promoting greater stability of themutant ¹⁰Fn3 at neutral pH as compared to the wild-type form (See, PCTPublication No. WO02/04523). A variety of additional alterations in the¹⁰Fn3 scaffold that are either beneficial or neutral have beendisclosed. See, for example, Batori et al., Protein Eng. 2002 December;15(12):1015-20; Koide et al., Biochemistry 2001 Aug. 28;40(34):10326-33.

Both variant and wild-type ¹⁰Fn3 proteins are characterized by the samestructure, namely seven beta-strand domain sequences designated Athrough G and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID NO: 1, the AB loopcorresponds to residues 15-16, the BC loop corresponds to residues21-30, the CD loop corresponds to residues 39-45, the DE loopcorresponds to residues 51-56, the EF loop corresponds to residues60-66, and the FG loop corresponds to residues 76-87 (Xu et al.,Chemistry & Biology 2002 9:933-942). The BC, DE and FG loops align alongone face of the molecule and the AB, CD and EF loops align along theopposite face of the molecule. In SEQ ID NO: 1, beta strand Acorresponds to residues 9-14, beta strand B corresponds to residues17-20, beta strand C corresponds to residues 31-38, beta strand Dcorresponds to residues 46-50, beta strand E corresponds to residues57-59, beta strand F corresponds to residues 67-75, and beta strand Gcorresponds to residues 88-94. The strands are connected to each otherthrough the corresponding loop, e.g., strands A and B are connected vialoop AB in the formation strand A, loop AB, strand B, etc. Residuesinvolved in forming the hydrophobic core (the “core amino acidresidues”) include the amino acids corresponding to the following aminoacids of SEQ ID NO: 1: L8, V10, A13, L18, I20, W22, Y32, I34, Y36, F48,V50, A57, I59, L62, Y68, I70, V72, A74, I88, 190 and Y92, wherein thecore amino acid residues are represented by the single letter amino acidcode followed by the position at which they are located within SEQ IDNO: 1. See e.g., Dickinson et al., J. Mol. Biol. 236: 1079-1092 (1994).

In some embodiments, the ¹⁰Fn3 polypeptide may be at least 40%, 50%,60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the human ¹⁰Fn3domain, shown in SEQ ID NO: 1. Much of the variability will generallyoccur in one or more of the loops. Each of the beta or beta-like strandsof a ¹⁰Fn3 polypeptide may consist essentially of an amino acid sequencethat is at least 80%, 85%, 90%, 95% or 100% identical to the sequence ofa corresponding beta or beta-like strand of SEQ ID NO: 1, provided thatsuch variation does not disrupt the stability of the polypeptide inphysiological conditions.

In some embodiments, the disclosure provides polypeptides comprising atenth fibronectin type III (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop, EF;and a loop, FG; and has at least one loop selected from loop BC, DE, andFG with an altered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain. In some embodiments, theBC and FG loops are altered. In some embodiments, the BC, DE, and FGloops are altered, i.e., the Fn3 domains comprise non-naturallyoccurring loops. By “altered” is meant one or more amino acid sequencealterations relative to a template sequence (corresponding humanfibronectin domain) and includes amino acid additions, deletions, andsubstitutions. Altering an amino acid sequence may be accomplishedthrough intentional, blind, or spontaneous sequence variation, generallyof a nucleic acid coding sequence, and may occur by any technique, forexample, PCR, error-prone PCR, or chemical DNA synthesis.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop. In some embodiments, the length of the loop may beextended by from 2-25 amino acids. In some embodiments, the length ofthe loop may be decreased by 1-11 amino acids. In particular, the FGloop of ¹⁰Fn3 is 12 residues long, whereas the corresponding loop inantibody heavy chains ranges from 4-28 residues. To optimize antigenbinding, therefore, the length of the FG loop of ¹⁰Fn3 may be altered inlength as well as in sequence to cover the CDR3 range of 4-28 residuesto obtain the greatest possible flexibility and affinity in antigenbinding.

In some embodiments, the polypeptide comprises a first Fn3 domain thatcomprises an amino acid sequence at least 80, 85, 90, 95, 98, or 100%identical to the non-loop regions of SEQ ID NO: 1, wherein at least oneloop selected from BC, DE, and FG is altered. In some embodiments, thepolypeptide comprises a second Fn3 domain that comprises an amino acidsequence at least 80, 85, 90, 95, 98, or 100% identical to the non-loopregions of SEQ ID NO: 1, wherein at least one loop selected from BC, DE,and FG is altered. In some embodiments, the altered BC loop has up to 10amino acid substitutions, up to 4 amino acid deletions, up to 10 aminoacid insertions, or a combination thereof. In some embodiments, thealtered DE loop has up to 6 amino acid substitutions, up to 4 amino aciddeletions, up to 13 amino acid insertions or a combination thereof. Insome embodiments, the FG loop has up to 12 amino acid substitutions, upto 11 amino acid deletions, up to 25 amino acid insertions or acombination thereof.

The ¹⁰Fn3 domains generally begin with amino acid number 1 of SEQ IDNO: 1. However, domains with amino acid deletions are also encompassedby the invention. In some embodiments, the first eight amino acids ofSEQ ID NO: 1 are deleted. Additional sequences may also be added to theN- or C-terminus. For example, an additional MG sequence may be placedat the N-terminus of an Fn3 domain, in particular at the N-terminus ofthe first Fn3 domain. The M will usually be cleaved off, leaving a G atthe N-terminus. In some embodiments, sequences may be placed at theC-terminus of the ¹⁰Fn3 domain, e.g., SEQ ID NOS: 17, 18, or 19. Inother embodiments, sequences placed at the C-terminus may be aC-terminally truncated fragment of SEQ ID NOs: 17, 18 or 19, including,for example, one of the following amino acid sequences (represented bythe single letter amino acid code): E, EI, EID, EIDKP (SEQ ID NO: 50),EIDKPS (SEQ ID NO: 51), or EIDKPC (SEQ ID NO: 52).

In some embodiments, the polypeptide comprises a first and/or second¹⁰Fn3 domain with a BC loop having the amino acid sequence of SEQ ID NO:2, a DE loop having the amino acid sequence of SEQ ID NO: 3, and an FGloop having the amino acid sequence of SEQ ID NO: 4, wherein the Fn3domain binds IGF-IR. In some embodiments, the polypeptide comprises afirst and/or second ¹⁰Fn3 domain with a BC loop having the amino acidsequence of SEQ ID NO: 5, a DE loop having the amino acid sequence ofSEQ ID NO: 6, and an FG loop having the amino acid sequence of SEQ IDNO: 7, wherein the Fn3 domain binds VEGFR2. In some embodiments, thepolypeptide comprises the amino acid sequence of any one of SEQ ID NOS:8-15, 29-31 and 63-70. In some embodiments, the polypeptide comprisesthe amino acid sequence at least 70, 75, 80, 85, 90, 95, or 100%identical to of any one of SEQ ID NOS: 8-15, 29-31 and 63-70.

Fibronectin naturally binds certain types of integrins through itsintegrin-binding motif, “arginine-glycine-aspartic acid” (RGD). In someembodiments, the polypeptide comprises a ¹⁰Fn3 domain that lacks the(RGD) integrin binding motif.

In one embodiment, a multivalent polypeptide comprises a polypeptidehaving the structure A-B-C, wherein A is a polypeptide comprising,consisting essentially of, or consisting of a ¹⁰Fn3 domain that binds toVEGFR2, B is a polypeptide linker, and C is a polypeptide comprising,consisting essentially of, or consisting of a ¹⁰Fn3 domain that binds toIGF-IR. In another embodiment, a multivalent polypeptide comprises apolypeptide having the structure A-B—C, wherein A is a polypeptidecomprising, consisting essentially of, or consisting of a ¹⁰Fn3 domainthat binds to IGF-IR, B is a polypeptide linker, and C is a polypeptidecomprising, consisting essentially of, or consisting of a ¹⁰Fn3 domainthat binds to VEGFR2. Specific examples of multivalent polypeptideshaving the structure A-B—C are polypeptides comprising (i) a polypeptidehaving an amino acid sequence set forth in any one of SEQ ID NOs: 8-15,29-31 and 63-70, or (ii) a polypeptide comprising an amino acid sequenceat least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of theamino acid sequences set forth in SEQ ID NOs: 8-15, 29-31 and 63-70.

In certain embodiments, the A or C region is a polypeptide comprising a¹⁰Fn3 domain that binds to VEGFR2, wherein the ¹⁰Fn3 domain has thestructure from N-terminus to C-terminus: beta strand A, loop AB, betastrand B, loop BC, beta strand C, loop CD, beta strand D, loop DE, betastrand E, loop EF, beta strand F, loop FG, beta strand G, wherein the BCloop has the amino acid sequence of SEQ ID NO: 5, the DE loop has theamino acid sequence of SEQ ID NO: 6, and the FG loop has the amino acidsequence of SEQ ID NO: 7, wherein the ¹⁰Fn3 domain folds into anantibody heavy chain variable region-like structure, and wherein thepolypeptide binds to VEGFR2 with a K_(D) of less than 100 nM. The ¹⁰Fn3domain that binds to VEGFR2 preferably folds into a structure whereinthe 7 beta strands are distributed between two beta sheets that packagainst each other forming a stable core and wherein the beta strandsare connected by the six loops which are solvent exposed. In exemplaryembodiments, the ¹⁰Fn3 domain is from 80-150 amino acids in length.

In certain embodiments, the A or C region is a 10Fn3 domain that bindsto VEGFR2 comprising a BC loop having the amino acid sequence of SEQ IDNO: 5, a DE loop having the amino acid sequence of SEQ ID NO: 6, and anFG loop having the amino acid sequence of SEQ ID NO: 7, wherein the¹⁰Fn3 domain binds to VEGFR2 with a K_(D) of less than 100 nM. Anexemplary VEGFR2 binder is represented by the sequence:

(SEQ ID NO: 47) EVVAATX_(n1) SLLIX_(a1) SWRHPHFPTRX_(a2) YYRITYGEX_(n2)QEFTVX_(a3) PLQPPTX_(a4) ATIX_(n3) DYTITVYAVX_(a5) TDGRNGRLLSIPX_(a6)ISINY RT.In SEQ ID NO: 47, the BC, DE and FG loops have a fixed sequence as shownin bold, the AB loop is represented by X_(n1), the CD is represented byX_(n2), and EF loop is represented by X_(n3), and the beta strands A-Gare underlined. X represents any amino acid and the subscript followingthe X represents an integer of the number of amino acids. In particular,n1 may be anywhere from 1-15, 2-15, 1-10, 2-10, 1-8, 2-8, 1-5, 2-5, 1-4,2-4, 1-3, 2-3, or 1-2 amino acids; n2 and n3 may each independently beanywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15,6-10, 6-8, 2-7, 5-7, or 6-7 amino acids; and a1-a6 may eachindependently comprise from 0-10, 0-5, 1-10, 1-5, or 2-5 amino acids. Inpreferred embodiments, n1 is 2 amino acids, n2 is 7 amino acids, n3 is 7amino acids, and a1-a6 is 0 amino acids. The sequences of the betastrands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, deletions or additions across all 7 scaffold regionsrelative to the corresponding amino acids shown in SEQ ID NO: 1. In anexemplary embodiment, the sequences of the beta strands may haveanywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to4, from 0 to 3, from 0 to 2, or from 0 to 1 conservative substitutionsacross all 7 scaffold regions relative to the corresponding amino acidsshown in SEQ ID NO: 1. In certain embodiments, the core amino acidresidues are fixed and any substitutions, conservative substitutions,deletions or additions occur at residues other than the core amino acidresidues. In certain embodiments, the VEGFR2 binder is represented bythe following amino acid sequence:

(SEQ ID NO: 40) EVVAATPTSLLI SWRHPHFPTR YYRITYGETGGNSPVQEFTV PL QPPTATISGLKPGVDYTITVYAV TDGRNGRLLSIP ISINYRT.In SEQ ID NO: 40, the sequence of the BC, DE and FG loops have a fixedsequence as shown in bold (e.g., a BC loop having the amino acidsequence of SEQ ID NO: 5, a DE loop having the amino acid sequence ofSEQ ID NO: 6, and an FG loop having the amino acid sequence of SEQ IDNO: 7) and the remaining sequence which is underlined (e.g., thesequence of the 7 beta strands and the AB, CD and EF loops) has anywherefrom 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, conservative substitutions, deletions or additionsrelative to the corresponding amino acids shown in SEQ ID NO: 35. Incertain embodiments, the core amino acid residues are fixed and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the core amino acid residues. The ¹⁰Fn3 domainthat binds to VEGFR2 may optionally comprise an N-terminal extension offrom 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids inlength. Exemplary N-terminal extensions include (represented by thesingle letter amino acid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 45),VSDVPRDL (SEQ ID NO: 46), and GVSDVPRDL (SEQ ID NO: 48), or N-terminaltruncations of any one of SEQ ID NOs: 45, 46 or 48. Other suitableN-terminal extensions include, for example, X_(n)SDVPRDL (SEQ ID NO:53), X_(n)DVPRDL (SEQ ID NO: 54), X_(n)VPRDL (SEQ ID NO: 55), X_(n)PRDL(SEQ ID NO: 56), X_(n)RDL (SEQ ID NO: 57), X_(a)DL (SEQ ID NO: 58), orX_(n)L, wherein n=0, 1 or 2 amino acids, wherein when n=1, X is Met orGly, and when n=2, X is Met-Gly. The ¹⁰Fn3 domain that binds to VEGFR2may optionally comprise a C-terminal tail. Exemplary C-terminal tailsinclude polypeptides that are from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3,1-2, or 1 amino acids in length. Specific examples of C-terminal tailsinclude EIDKPSQ (SEQ ID NO: 17), EIDKPCQ (SEQ ID NO: 18), and EIDK (SEQID NO: 19). In other embodiments, suitable C-terminal tails may be aC-terminally truncated fragment of SEQ ID NOs: 17, 18 or 19, including,for example, one of the following amino acid sequences (represented bythe single letter amino acid code): E, EI, EID, EIDKP (SEQ ID NO: 50),EIDKPS (SEQ ID NO: 51), or EIDKPC (SEQ ID NO: 52). Other suitableC-terminal tails include, for example, ES, EC, EGS, EGC, EGSGS (SEQ IDNO: 71), or EGSGC (SEQ ID NO: 72). In certain embodiments, the ¹⁰Fn3domain that binds to VEGFR2 comprises both an N-terminal extension and aC-terminal tail. In exemplary embodiments, the A region comprises anN-terminal extension beginning with Gly or Met-Gly and a C-terminalextension that does not contain a cysteine residue and the B regioncomprises an N-terminal extension that does not start with a Met and aC-terminal extension that comprises a cysteine residue. Specificexamples of ¹⁰Fn3 domains that bind to VEGFR2 are polypeptidescomprising (i) a polypeptide having an amino acid sequence set forth inany one of SEQ ID NOs: 16, 28 and 40-44, or (ii) a polypeptidecomprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or99% identical to the amino acid sequence set forth in any one of SEQ IDNOs: 16, 28 and 40-44.

In certain embodiments, the A or C region is a polypeptide comprising a¹⁰Fn3 domain that binds to IGF-IR, wherein the ¹⁰Fn3 domain has thestructure from N-terminus to C-terminus: beta strand A, loop AB, betastrand B, loop BC, beta strand C, loop CD, beta strand D, loop DE, betastrand E, loop EF, beta strand F, loop FG, beta strand G, wherein the BCloop has the amino acid sequence of SEQ ID NO: 2, the DE loop has theamino acid sequence of SEQ ID NO: 3, and the FG loop has the amino acidsequence of SEQ ID NO: 4, wherein the ¹⁰Fn3 domain folds into anantibody heavy chain variable region-like structure, and wherein thepolypeptide binds to IGF-IR with a K_(D) of less than 100 nM. The ¹⁰Fn3domain that binds to IGF-IR preferably folds into a structure whereinthe 7 beta strands are distributed between two beta sheets that packagainst each other forming a stable core and wherein the beta strandsare connected by the six loops which are solvent exposed. In exemplaryembodiments, the ¹⁰Fn3 domain is from 80-150 amino acids in length.

In certain embodiments, the A or C region is a ¹⁰Fn3 domain that bindsto IGF-IR comprising a BC loop having the amino acid sequence of SEQ IDNO: 2, a DE loop having the amino acid sequence of SEQ ID NO: 3, and anFG loop having the amino acid sequence of SEQ ID NO: 4, wherein the¹⁰Fn3 domain binds to IGF-IR with a K_(D) of less than 100 nM. Anexemplary IGF-IR binder is represented by the sequence:

(SEQ ID NO: 49) EVVAATX_(n1) SLLIX_(a1) SWSARLKVARX_(a2) YYRITYGEX_(n2)QEFTVX_(a3) PKNVYTX_(a4) ATIX_(n3) DYTITVYAVX_(a5) TRFRDYQPX_(a6)ISINYRT.In SEQ ID NO: 49, the BC, DE and FG loops have a fixed sequence as shownin bold, the AB loop is represented by X_(n1), the CD loop isrepresented by X_(n2), and the EF loop is represented by X_(n3), and thebeta strands A-G are underlined. X represents any amino acid and thesubscript following the X represents an integer of the number of aminoacids. In particular, n1 may be anywhere from 1-15, 2-15, 1-10, 2-10,1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3, or 1-2 amino acids; n2 and n3may each independently be anywhere from 2-20, 2-15, 2-10, 2-8, 5-20,5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids;and a1-a6 may each independently comprise from 0-10, 0-5, 1-10, 1-5, or2-5 amino acids. In preferred embodiments, n1 is 2 amino acids, n2 is 7amino acids, n3 is 7 amino acids, and a1-a6 is 0 amino acids. Thesequences of the beta strands may have anywhere from 0 to 10, from 0 to8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, orfrom 0 to 1 substitutions, deletions or additions across all 7 scaffoldregions relative to the corresponding amino acids shown in SEQ ID NO: 1.In an exemplary embodiment, the sequences of the beta strands may haveanywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to4, from 0 to 3, from 0 to 2, or from 0 to 1 conservative substitutionsacross all 7 scaffold regions relative to the corresponding amino acidsshown in SEQ ID NO: 1. In certain embodiments, the core amino acidresidues are fixed and any substitutions, conservative substitutions,deletions or additions occur at residues other than the core amino acidresidues. In certain embodiments, the IGF-IR binder is represented bythe following amino acid sequence:

(SEQ ID NO: 40) EVVAATPTSLLI SWSARLKVAR YYRITYGETGGNSPVQEFTV PK NVYTATISGLKPGVDYTITVYAV TRFRDYQP ISINYRT.In SEQ ID NO: 40, the sequence of the BC, DE and FG loops have a fixedsequence as shown in bold (e.g., a BC loop having the amino acidsequence of SEQ ID NO: 2, a DE loop having the amino acid sequence ofSEQ ID NO: 3, and an FG loop having the amino acid sequence of SEQ IDNO: 4) and the remaining sequence which is underlined (e.g., thesequence of the 7 beta strands and the AB, CD and EF loops) has anywherefrom 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, conservative substitutions, deletions or additionsrelative to the corresponding amino acids shown in SEQ ID NO: 40. Incertain embodiments, the core amino acid residues are fixed and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the core amino acid residues. The ¹⁰Fn3 domainthat binds to IGF-IR may optionally comprise an N-terminal extension offrom 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids inlength. Exemplary N-terminal extensions include (represented by thesingle letter amino acid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 45),VSDVPRDL (SEQ ID NO: 46), and GVSDVPRDL (SEQ ID NO: 48), or N-terminallytruncated fragments of any one of SEQ ID NOs: 45, 46 or 48. Othersuitable N-terminal extensions include, for example, X_(n)SDVPRDL (SEQID NO: 53), X_(n)DVPRDL (SEQ ID NO: 54), X_(n)VPRDL (SEQ ID NO: 55),X_(n)PRDL (SEQ ID NO: 56), X_(n)RDL (SEQ ID NO: 57), X_(n)DL (SEQ ID NO:58), or X_(n)L, wherein n=0, 1 or 2 amino acids, wherein when n=1, X isMet or Gly, and when n=2, X is Met-Gly. The ¹⁰Fn3 domain that binds toIGF-IR may optionally comprise a C-terminal tail. Exemplary C-terminaltails include polypeptides that are from 1-20, 1-15, 1-10, 1-8, 1-5,1-4, 1-3, 1-2, or 1 amino acids in length. Specific examples ofC-terminal tails include EIDKPSQ (SEQ ID NO: 17), EIDKPCQ (SEQ ID NO:18), and EIDK (SEQ ID NO: 19). In other embodiments, suitable C-terminaltails may be a C-terminally truncated fragment of SEQ ID NOs: 17, 18 or19, including, for example, one of the following amino acid sequences(represented by the single letter amino acid code): E, EI, EID, EIDKP(SEQ ID NO: 50), EIDKPS (SEQ ID NO: 51), or EIDKPC (SEQ ID NO: 52).Other suitable C-terminal tails include, for example, ES, EC, EGS, EGC,EGSGS (SEQ ID NO: 71), or EGSGC (SEQ ID NO: 72). In certain embodiments,the ¹⁰Fn3 domain that binds to IGF-IR comprises both an N-terminalextension and a C-terminal tail. In exemplary embodiments, the A regioncomprises an N-terminal extension beginning with Gly or Met-Gly and aC-terminal extension that does not contain a cysteine residue and the Bregion comprises an N-terminal extension that does not start with a Metand a C-terminal extension that comprises a cysteine residue. Specificexamples of ¹⁰Fn3 domains that bind to IGF-IR are polypeptidescomprising (i) a polypeptide having the amino acid sequence set forth inany one of SEQ ID NOs: 26-27 and 35-39, or (ii) a polypeptide comprisingan amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99%identical to the amino acid sequence set forth in any one of SEQ ID NOs:26-27 and 35-39.

The B region is a polypeptide linker. Exemplary polypeptide linkersinclude polypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3,or 1-2 amino acids. Specific examples of suitable polypeptide linkersare described further herein. In certain embodiments, the linker may bea C-terminal tail polypeptide as described herein, an N-terminalextension polypeptide as described herein, or a combination thereof.

In one embodiment, a multivalent polypeptide comprises a polypeptidehaving the structure X₁-A-X₂—B—X₃—C—X₄, wherein X₁ is an optionalN-terminal extension, A is a ¹⁰Fn3 domain that binds to VEGFR2, X₂ is anoptional C-terminal tail, B is a polypeptide linker, X₃ is an optionalN-terminal extension, C is a ¹⁰Fn3 domain that binds to IGF-IR, and X₄is an optional C-terminal tail. In another embodiment, a multivalentpolypeptide comprises a polypeptide having the structureX₁-A-X₂—B—X₃—C—X₄, wherein X₁ is an optional N-terminal extension, A isa ¹⁰Fn3 domain that binds to IGF-IR, X₂ is an optional C-terminal tail,B is a polypeptide linker, X₃ is an optional N-terminal extension, C isa ¹⁰Fn3 domain that binds to VEGFR2, and X₄ is an optional C-terminaltail. Specific examples of suitable N-terminal extensions and C-terminaltails are described above. In certain embodiments, one or more of X₁,X₂, B, X₃ or X₄ may comprise an amino acid residue suitable forpegylation, such as a cysteine or lysine residue. In exemplaryembodiments, X₄ comprises at least one amino acid suitable forpegylation, such as a cysteine or lysine residue. Specific examples ofsuitable polypeptide linkers are described further below. Specificexamples of multivalent polypeptides having the structureX₁-A-X₂—B—X₃—C—X₄ are polypeptides comprising (i) a polypeptide havingthe amino acid sequence set forth in any one of SEQ ID NOs: 8-15, 29-31and 63-70, or (ii) a polypeptide comprising an amino acid sequence atleast 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence set forth in any one of SEQ ID NOs: 8-15, 29-31 and 63-70.

In exemplary embodiments, X as defined herein is a naturally occurringamino acid.

Polypeptide Linkers

The application provides multivalent polypeptides comprising at leasttwo Fn3 domains linked via a polypeptide linker. The polypeptidescomprise an N-terminal domain comprising a first Fn3 domain and aC-terminal domain comprising a second Fn3 domain. The first and secondFn3 domains may be directly or indirectly linked via a polypeptidelinker. Additional linkers or spacers, e.g., SEQ ID NOS: 17, 18, or 19,may be introduced at the C-terminus of the first Fn3 domain between theFn3 domain and the polypeptide linker. Additional linkers or spacers maybe introduced at the N-terminus of the second Fn3 domain between the Fn3domain and the polypeptide linker.

Suitable linkers for joining the Fn3 domains are those which allow theseparate domains to fold independently of each other forming a threedimensional structure that permits high affinity binding to a targetmolecule. The application provides that suitable linkers that meet theserequirements comprise glycine-serine based linkers, glycine-prolinebased linkers, proline-alanine based linkers as well as the linker SEQID NO: 20. The Examples described herein demonstrate that Fn3 domainsjoined via these linkers retain their target binding function. In someembodiments, the linker is a glycine-serine based linker. These linkerscomprise glycine and serine residues and may be between 8 and 50, 10 and30, and 10 and 20 amino acids in length. Examples of such linkersinclude SEQ ID NOS: 23, 24, and 25. In some embodiments the polypeptidelinker is selected from SEQ ID NOS: 21 and 22. In some embodiments, thelinker is a glycine-proline based linker. These linkers comprise glycineand proline residues and may be between 3 and 30, 10 and 30, and 3 and20 amino acids in length. Examples of such linkers include SEQ ID NOS:32, 33, and 34. In some embodiments, the linker is a proline-alaninebased linker. These linkers comprise proline and alanine residues andmay be between 3 and 30, 10 and 30, 3 and 20 and 6 and 18 amino acids inlength. Examples of such linkers include SEQ ID NOS: 60, 61 and 62. Itis contemplated, that the optimal linker length and amino acidcomposition may be determined by routine experimentation by methods wellknown in the art. In some embodiments, the polypeptide linker is SEQ IDNO: 20.

Pharmacokinetic Moieties

In one aspect, the application provides for multivalent polypeptidesfurther comprising a pharmacokinetic (PK) moiety. Improvedpharmacokinetics may be assessed according to the perceived therapeuticneed. Often it is desirable to increase bioavailability and/or increasethe time between doses, possibly by increasing the time that a proteinremains available in the serum after dosing. In some instances, it isdesirable to improve the continuity of the serum concentration of theprotein over time (e.g., decrease the difference in serum concentrationof the protein shortly after administration and shortly before the nextadministration). The polypeptides may be attached to a moiety thatreduces the clearance rate of the polypeptide in a mammal (e.g., mouse,rat, or human) by greater than three-fold relative to the unmodifiedpolypeptide. Other measures of improved pharmacokinetics may includeserum half-life, which is often divided into an alpha phase and a betaphase. Either or both phases may be improved significantly by additionof an appropriate moiety.

Moieties that tend to slow clearance of a protein from the blood, hereinreferred to as “PK moieties”, include polyoxyalkylene moieties, e.g.,polyethylene glycol, sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc, Fc fragments, transferrin, or serumalbumin). The polypeptides may be fused to albumin or a fragment(portion) or variant of albumin as described in U.S. Publication No.20070048282.

In some embodiments, the PK moiety is a serum albumin binding proteinsuch as those described in U.S. Publication Nos. 2007/0178082 and2007/0269422.

In some embodiments, the PK moiety is a serum immunoglobulin bindingprotein such as those described in U.S. Publication No. 2007/0178082.

In some embodiments, the multivalent polypeptide comprises polyethyleneglycol (PEG). One or more PEG molecules may be attached at differentpositions on the protein, and such attachment may be achieved byreaction with amines, thiols or other suitable reactive groups. Theamine moiety may be, for example, a primary amine found at theN-terminus of a polypeptide or an amine group present in an amino acid,such as lysine or arginine. In some embodiments, the PEG moiety isattached at a position on the polypeptide selected from the groupconsisting of: a) the N-terminus; b) between the N-terminus and the mostN-terminal beta strand or beta-like strand; c) a loop positioned on aface of the polypeptide opposite the target-binding site; d) between theC-terminus and the most C-terminal beta strand or beta-like strand; ande) at the C-terminus.

Pegylation may be achieved by site-directed pegylation, wherein asuitable reactive group is introduced into the protein to create a sitewhere pegylation preferentially occurs. In some embodiments, the proteinis modified to introduce a cysteine residue at a desired position,permitting site directed pegylation on the cysteine. In someembodiments, the polypeptide comprises a Cys containing linker such asSEQ ID NO: 18, which permits site directed pegylation. In someembodiments, the Cys containing linker is introduced at the 3′ end ofthe second Fn3 domain (i.e., the domain most C-terminal in thepolypeptide). PEG may vary widely in molecular weight and may bebranched or linear.

In some embodiments, the multivalent polypeptide comprises an Fn3 domainand a PK moiety. In some embodiments, the Fn3 domain is a ¹⁰Fn3 domain.In some embodiments, the PK moiety increases the serum half-life of thepolypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3 domainalone.

In some embodiments, the PK moiety is linked to the Fn3 domain via atleast one disulfide bond, a peptide bond, a polypeptide, a polymericsugar, or a polyethylene glycol moiety. Exemplary polypeptide linkersinclude PSTSTST (SEQ ID NO: 20), EIDKPSQ (SEQ ID NO: 17), and GSlinkers, such as GSGSGSGSGS (SEQ ID NO: 21) and multimers thereof. Insome embodiments the PK moiety is human serum albumin. In someembodiments, the PK moiety is transferrin.

Target

The application provides multivalent polypeptides comprising a first Fn3domain that binds to a first target molecule and a second Fn3 domainthat binds to a second target molecule. The first and second targetmolecules may be the same or different target molecules. When the firstand second target molecules are the same, the Fn3 domains, i.e., thebinding loops, may be the same or different. Therefore, the first andsecond Fn3 domains may bind to the same target but at differentepitopes. By introducing sequence variation in the loop regions, inparticular loops BC, DE, and FG, Fn3 domains can be generated that canbind to almost any target molecule.

Polypeptide binding to a target molecule may be assessed in terms ofequilibrium constants (e.g., dissociation, K_(D)) and in terms ofkinetic constants (e.g., on rate constant, k_(on) and off rate constant,k_(off)). An Fn3 domain will generally bind to a target molecule with aK_(D) of less than 500 nM, 100 nM, 1 nM, 500 pM, 100 pM or less,although higher K_(D) values may be tolerated where the k_(off) issufficiently low or the k_(on) is sufficiently high.

In some embodiments, the first and/or second Fn3 domain binds a targetselected from IGF-IR, FGFR, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, c-Kit,human p185 receptor-like tyrosine kinase, EGFR, HER2, HER3, HER4, c-Met,folate receptor, PDGFR, VEGFR1, VEGFR2, VEGFR3, human vascularendothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, human CD20, humanCD18, human CD11a, human apoptosis receptor-2 (Apo-2), human alpha4beta7integrin, human GPIIb-IIIa integrin, stem cell factor (SCF), human CD3,IGF-IR, Ang1, Ang2, fibroblast growth factor, epidermal growth factor,hepatocyte growth factor, or Tie2.3. In some embodiments, the first Fn3domain is in the N-terminal position and binds IGF-IR and the second Fn3domain binds VEGFR2. In some embodiments, the first Fn3 domain is in theN-terminal position and binds VEGFR2 and the second domain binds IGF-IR.In some embodiments, the first Fn3 domain is in the N-terminal positionand binds IGF-1R and the second domain binds VEGFR2.

In certain embodiments, the multivalent polypeptides comprise first andsecond Fn3 domains that bind to different targets. In such embodiments,it may be desirable to tune the potency of one Fn3 binding domainrelative to the other Fn3 binding domain. For example, if the bindingaffinity of the first Fn3 domain is significantly higher than thebinding affinity of the second Fn3 domain, the biological effect of thefirst Fn3 domain could overwhelm the effects of the second of second Fn3domain. Accordingly, in certain embodiments, it may be desirable for thebinding affinities of the first and second Fn3 domains of a multivalentpolypeptide to be similar to each other, e.g., binding affinities within100-fold, 30-fold, 10-fold, 3-fold, 1-fold, 0.3-fold or 0.1-fold, ofeach other, or binding affinities within 0.1-fold to 10-fold, within0.3-fold to 10-fold, within 0.1-fold to 3-fold, within 0.3-fold to3-fold, within 0.1-fold to 1-fold, within 0.3-fold to 1-fold, within1-fold to 10-fold, within 3-fold to 10-fold, within 3-fold to 30-fold,or within 1-fold to 3-fold of each other.

Multi-Domain Embodiments

One aspect of the application provides for multivalent polypeptidesfurther comprising a binding moiety. In some embodiments, the bindingmoiety binds a human target protein with a K_(D) of less than 10⁻⁶M,10⁻⁷M, 10⁻⁸M, 10⁻⁹M, or 10⁻⁹M.

In some embodiments, the binding moiety binds a tumor associated targetor antigen. In some embodiments antigen targeting will help localize themultivalent polypeptide in terms of tissue distribution or increasedlocal concentration affect either in the tissue or desired cell type.

In some embodiments, the binding moiety binds a tumor associated targetor antigen, such as, for example, carbonic anhydrase IX, A3, antigenspecific for A33 antibody, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15,CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80,HLA-DR, NCA95, NCA90, HCG and its subunits, CEA (CEACAM-5), CEACAM-6,CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, hypoxia induciblefactor (HIF), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophageinhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4-antigen,PSA, PSMA, RS5, S100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growthfactor-I (IGF-I), insulin growth factor-II (IGF-II), Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, placenta growthfactor (P1GF), 17-1A-antigen, an angiogenesis marker (e.g., ED-Bfibronectin), an oncogene marker, an oncogene product, and othertumor-associated antigens. Recent reports on tumor associated antigensinclude Mizukami et al., (2005, Nature Med. 11:992-97); Hatfield et al.,(2005, Curr. Cancer Drug Targets 5:229-48); Vallbohmer et al. (2005, J.Clin. Oncol. 23:3536-44); and Ren et al. (2005, Ann. Surg. 242:55-63),each incorporated herein by reference.

In some embodiments, the binding moiety is selected from an antibodymoiety. An antibody moiety refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. As such, the term antibody moiety encompasses not onlywhole antibody molecules, but also antibody multimers and antibodyfragments as well as variants (including derivatives) of antibodies,antibody multimers and antibody fragments. Examples of antibody moietiesinclude, but are not limited to single chain Fvs (scFvs), Fab fragments,Fab′ fragments, F(ab′)2, disulfide linked Fvs (sdFvs), and Fvs. Antibodymoieties may be, for example, monoclonal, chimeric, human, or humanized.

In some embodiments, the antibody moiety is selected from (i) a Fabfragment, having VL, CL, VH and CH1 domains; (ii) a Fab′ fragment, whichis a Fab fragment having one or more cysteine residues at the C-terminusof the CH1 domain; (iii) a Fd fragment having VH and CH1 domains; (iv) aFd′ fragment having VH and CH1 domains and one or more cysteine residuesat the C-terminus of the CH1 domain; (v) a Fv fragment having the VL andVH domains of a single arm of an antibody; (vi) a dAb fragment (Ward etal., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii)isolated CDR regions; (viii) F(ab′)2 fragments, a bivalent fragmentincluding two Fab′ fragments linked by a disulphide bridge at the hingeregion; (ix) single chain antibody molecules (e.g., single chain Fv;scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS(USA) 85:5879-5883 (1988)); (x) a “diabody” with two antigen bindingsites, comprising a heavy chain variable domain (VH) connected to alight chain variable domain (VL) in the same polypeptide chain (see,e.g., EP Patent Publication No. 404,097; WO93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and (xi) a“linear antibody” comprising a pair of tandem Fd segments(VH-CH1-VH—CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

In some embodiments, an antibody moiety is a single domain antibody.Examples include, but are not limited to, heavy chain antibodies,antibodies naturally devoid of light chains, single domain antibodiesderived from conventional 4-chain antibodies, engineered antibodies andsingle domain scaffolds other than those derived from antibodies. Singledomain antibodies may be derived from any species including, but notlimited to mouse, human, camel, llama, goat, rabbit, bovine.

In some embodiments, a single domain antibody is a naturally occurringsingle domain antibody such as VHH domains. VHHs are heavy chainvariable domains derived from immunoglobulins naturally devoid of lightchains such as those derived from Camelidae (including camel, dromedary,llama, vicuna, alpaca and guanaco) as described in WO94/04678. VHHmolecules are about 10 times smaller than IgG molecules. Since VHH's areknown to bind to ‘unusual’ epitopes such as cavities or grooves, theaffinity of such VHH's may be more suitable for therapeutic treatment,PCT Publication No. WO97/49805.

In some embodiments, the single domain antibody is a VHH that binds aserum protein as described in U.S. Publication No. 20070178082. Theserum protein may be any suitable protein found in the serum of subject,or fragment thereof. In some embodiments, the serum protein is serumalbumin, serum immunoglobulins, thyroxine-binding protein, transferrin,or fibrinogen.

Various techniques have been developed for the production of antibodyfragments that may be used to make antibody fragments used in theinvention. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)2 fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)2 fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. The antibody fragmentmay also be a “linear antibody”, e.g., as described in U.S. Pat. No.5,641,870 for example. Such linear antibody fragments may bemonospecific or bispecific.

In some embodiments, the binding moiety comprises one or more avimersequences. Avimers were developed from human extracellular receptordomains by in vitro exon shuffling and phage display. (Silverman et al.,2005, Nat. Biotechnol. 23:1493-94; Silverman et al., 2006, Nat.Biotechnol. 24:220.) The resulting multidomain proteins may comprisemultiple independent binding domains that may exhibit improved affinity(in some cases sub-nanomolar) and specificity compared withsingle-epitope binding proteins. Additional details concerning methodsof construction and use of avimers are disclosed, for example, in U.S.Patent Publication Nos. 20040175756, 20050048512, 20050053973,20050089932 and 20050221384, which is incorporated herein by referencein their entirety.

In some embodiments, the binding moiety comprises one or more lipocalinrelated sequences, e.g., anticalins or lipocalin derivatives. Anticalinsor lipocalin derivatives are a type of binding proteins that haveaffinities and specificities for various target molecules, includingthose described herein. Such proteins are described in US PatentPublication Nos. 20060058510, 20060088908, 20050106660, and PCTPublication No. WO2006/056464.

In some embodiments, the binding moiety comprises one or moretetranectin C-type lectin related sequences or trinectins, e.g.,tetranectin C-type lectin or tetranectin C-type lectin derivatives.Tetranectin C-type lectins or tetranectin C-type lectin derivatives area type of binding proteins that have affinities and specificities forvarious target molecules including those described herein. Differenttetranectin C-type lectin and related proteins are described in PCTPublication Nos. WO2006/053568, WO2005/080418, WO2004/094478,WO2004/039841, WO2004/005335, WO2002/048189, WO98/056906, and U.S.Patent Publication No. 20050202043.

In some embodiments, the binding moiety comprises one or more naturalankyrin repeat proteins, e.g., DARPins (Molecular Partners).

In some embodiments, the binding moiety comprises one or moreAffibodies™. Affibodies™ are derived from the IgG binding domain ofStaphyloccal Protein A. Novel binding properties can be achieved byaltering residues located near the binding surface of the Protein Adomain.

In some embodiments, the binding moiety comprises one or more cysteineknot based protein scaffolds, i.e., microbodies (Selecore/NascaCell).

In some embodiments, the binding moiety comprises one or moreTrans-Bodies™. Trans-Bodies™ are based on transferrin scaffolds(BioResis/Pfizer).

In some embodiments, the binding moiety comprises binding proteins basedon gamma-crystalline or ubiquitin. These so-called Affilin™ (ScilProteins) molecules are characterized by the de novo design of a bindingregion in beta sheet structures of the proteins. Affilin™ molecules havebeen described in U.S. Publication No. 20070248536.

Conjugation

The multivalent polypeptide and the binding moiety may be linked via atleast one disulfide bond, a peptide bond, a polypeptide, a polymericsugar, or a PEG moiety.

In some embodiments, the multivalent polypeptide and the binding moietyare linked via a polypeptide. In some embodiments, the polypeptidelinker is SEQ ID NOS: 17, 18, or 20.

In some embodiments, the multivalent polypeptide and the binding moietyare linked via a polypeptide linker having a protease site that iscleavable by a protease in the blood or target tissue. Such embodimentscan be used to release two or more therapeutic proteins for betterdelivery or therapeutic properties or more efficient production comparedto separately producing such proteins.

In some embodiments, the multivalent polypeptide and the binding moietyare linked via a biocompatible polymer such as a polymeric sugar. Suchpolymeric sugar can include an enzymatic cleavage site that is cleavableby an enzyme in the blood or target tissue. Such embodiments can be usedto release two or more therapeutic proteins for better delivery ortherapeutic properties or more efficient production compared toseparately producing such proteins.

In some embodiments, the multivalent polypeptide and the binding moietyare linked via a polymeric linker. Polymeric linkers can be used tooptimally vary the distance between each protein moiety to create aprotein with one or more of the following characteristics: 1) reduced orincreased steric hindrance of binding of one or more protein domain whenbinding to a protein of interest, 2) increased protein stability orsolubility without searching for additional amino acid substitutions toincrease stability or solubility (e.g., solubility at least about 20mg/ml, or at least about 50 mg/ml), 3) decreased protein aggregationwithout searching for additional amino acid substitutions to decreasestability (e.g., as measured by SEC), and 4) increased the overallavidity or affinity of the protein by adding additional binding domains.

In some embodiments, the multivalent polypeptide comprises a second¹⁰Fn3 domain comprising the linker of SEQ ID NO: 18. PEG is conjugatedto the cysteine moiety in the linker sequence and links the multivalentpolypeptide and the binding moiety.

PEGylated Embodiments

One aspect of the application provides linking the multivalentpolypeptides to nonproteinaceous polymers. In some embodiments, thepolymer is polyethylene glycol (“PEG”), polypropylene glycol, orpolyoxyalkylenes, as described in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. In some embodiments, themultivalent polypeptides comprise an Fn3 domain. In some embodiments,the polymer is a PEG moiety. In addition, the application provides N orC terminal PEG conjugation to antibody moieties (e.g., camel antibodiesand their derivatives, as well as single chain and domain antibodies;and particularly those expressed from microbes) and antibody-likemoieties (e.g., derivatives of lipocalins, ankyrins, multiple Cys-Cysdomains, and tetranectins; and particularly those expressed frommicrobes).

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula: X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH(1), where n is 20 to 2300 and X is H or a terminal modification, e.g.,a C₁₋₄ alkyl. In one embodiment, the PEG of the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). APEG can contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462. One form of PEGsincludes two PEG side-chains (PEG2) linked via the primary amino groupsof a lysine (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol.Chem., 252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a binding polypeptide containing a PEGmolecule is also known as a conjugated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated.

The size of PEG utilized will depend on several factors including theintended use of the multivalent polypeptide. Larger PEGs are preferredto increase half life in the body, blood, non-blood extracellular fluidsor tissues. For in vivo cellular activity, PEGs of the range of about 10to 60 kDa are preferred, as well as PEGs less than about 100 kDa andmore preferably less than about 60 kDa, though sizes greater than about100 kDa can be used as well. For in vivo imaging application, smallerPEGs, generally less than about 20 kDa, may be used that do not increasehalf life as much as larger PEGs so as to permit quicker distributionand less half life. A variety of molecular mass forms of PEG can beselected, e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to2300), for conjugating to binding polypeptides of the invention. Thenumber of repeating units “n” in the PEG is approximated for themolecular mass described in Daltons. It is preferred that the combinedmolecular mass of PEG on an activated linker is suitable forpharmaceutical use. Thus, in one embodiment, the molecular mass of thePEG molecules does not exceed 100,000 Da. For example, if three PEGmolecules are attached to a linker, where each PEG molecule has the samemolecular mass of 12,000 Da (each n is about 270), then the totalmolecular mass of PEG on the linker is about 36,000 Da (total n is about820). The molecular masses of the PEG attached to the linker can also bedifferent, e.g., of three molecules on a linker two PEG molecules can be5,000 Da each (each n is about 110) and one PEG molecule can be 12,000Da (n is about 270). In some embodiments, one PEG moiety is conjugatedto the multivalent polypeptide. In some embodiments, the PEG moiety isabout 20, 30, 40, 50, 60, 70, 80, or 90 KDa. In some embodiments, thePEG moiety is about 40 KDa.

In some embodiments, PEGylated multivalent polypeptides contain one, twoor more PEG moieties. In one embodiment, the PEG moiety(ies) are boundto an amino acid residue which is on the surface of the protein and/oraway from the surface that contacts the target ligand. In oneembodiment, the combined or total molecular mass of PEG in PEG-bindingpolypeptide is from about 3,000 Da to 60,000 Da, or from about 10,000 Dato 36,000 Da. In a one embodiment, the PEG in pegylated bindingpolypeptide is a substantially linear, straight-chain PEG.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated binding polypeptide will be usedtherapeutically, the desired dosage, circulation time, resistance toproteolysis, immunogenicity, and other considerations. For a discussionof PEG and its use to enhance the properties of proteins, see N. V.Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993).

In some embodiments, a multivalent polypeptide is covalently linked toone poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, a binding polypeptide'sε-amino group of a lysine is the available (free) amino group.

In one specific embodiment, carbonate esters of PEG are used to form thePEG-binding polypeptide conjugates. N,N′-disuccinimidylcarbonate (DSC)may be used in the reaction with PEG to form active mixedPEG-succinimidyl carbonate that may be subsequently reacted with anucleophilic group of a linker or an amino group of a bindingpolypeptide (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similartype of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of a multivalent polypeptide can be performed according tothe methods of the state of the art, for example by reaction of thebinding polypeptide with electrophilically active PEGs (supplier:Shearwater Corp., USA, www.shearwatercorp.com). Preferred PEG reagentsof the present invention are, e.g., N-hydroxysuccinimidyl propionates(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., et al.,Bioconjugate Chem. 6 (1995) 62-69). Such methods may used to pegylatedat a ε-amino group of a binding polypeptide lysine or the N-terminalamino group of the binding polypeptide.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a binding polypeptide (Sartore, L., et al., Appl. Biochem.Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368(1996); Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat. No.5,766,897). U.S. Pat. Nos. 6,610,281 and 5,766,897 describes exemplaryreactive PEG species that may be coupled to sulfhydryl groups.

In some embodiments, the pegylated a multivalent polypeptide is producedby site-directed pegylation, particularly by conjugation of PEG to acysteine moiety at the N- or C-terminus. In some embodiments, themultivalent polypeptide is an Fn3 domain covalently bound to a PEGmoiety, wherein at least one of the loops of said Fn3 domainparticipates in target binding. The PEG moiety may be attached to theFn3 polypeptide by site directed pegylation, such as by attachment to aCys residue, where the Cys residue may be positioned at the N-terminusof the Fn3 polypeptide or between the N-terminus and the most N-terminalbeta or beta-like strand or at the C-terminus of the Fn3 polypeptide orbetween the C-terminus and the most C-terminal beta or beta-like strand.A Cys residue may be situated at other positions as well, particularlyany of the loops that do not participate in target binding. A PEG moietymay also be attached by other chemistry, including by conjugation toamines.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a binding polypeptide, the cysteine residues are native tothe binding polypeptide, whereas in other embodiments, one or morecysteine residues are engineered into the binding polypeptide. Mutationsmay be introduced into a binding polypeptide coding sequence to generatecysteine residues. This might be achieved, for example, by mutating oneor more amino acid residues to cysteine. Preferred amino acids formutating to a cysteine residue include serine, threonine, alanine andother hydrophilic residues. Preferably, the residue to be mutated tocysteine is a surface-exposed residue. Algorithms are well-known in theart for predicting surface accessibility of residues based on primarysequence or a protein. Alternatively, surface residues may be predictedby comparing the amino acid sequences of binding polypeptides, giventhat the crystal structure of the framework based on which bindingpolypeptides are designed and evolved has been solved (see Himanen etal., Nature. (2001) 20-27; 414(6866):933-8) and thus the surface-exposedresidues identified. In one embodiment, cysteine residues are introducedinto binding polypeptides at or near the N- and/or C-terminus, or withinloop regions. Pegylation of cysteine residues may be carried out using,for example, PEG-maleiminde, PEG-vinylsulfone, PEG-iodoacetamide, orPEG-orthopyridyl disulfide.

In some embodiments, the pegylated binding polypeptide comprises a PEGmolecule covalently attached to the alpha amino group of the N-terminalamino acid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) JPET, 297,1059, and U.S. Pat. No. 5,824,784. Theuse of a PEG-aldehyde for the reductive amination of a protein utilizingother available nucleophilic amino groups is described in U.S. Pat. No.4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254,12579, and inChamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated binding polypeptide comprises one ormore PEG molecules covalently attached to a linker, which in turn isattached to the alpha amino group of the amino acid residue at theN-terminus of the binding polypeptide. Such an approach is disclosed inU.S. Publication No. 2002/0044921 and PCT Publication No. WO94/01451.

In one embodiment, a binding polypeptide is pegylated at the C-terminus.In a specific embodiment, a protein is pegylated at the C-terminus bythe introduction of C-terminal azido-methionine and the subsequentconjugation of a methyl-PEG-triarylphosphine compound via the Staudingerreaction. This C-terminal conjugation method is described in Cazalis etal., C-Terminal Site-Specific PEGylation of a Truncated ThrombomodulinMutant with Retention of Full Bioactivity, Bioconjug Chem. 2004;15(5):1005-1009.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g., gel filtration) and ion exchange chromatography. Products mayalso be separated using SDS-PAGE. Products that may be separated includemono-, di-, tri-poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment of the invention, the PEG in a pegylated multivalentpolypeptide is not hydrolyzed from the pegylated amino acid residueusing a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8to 16 hours at room temperature, and is thus stable. In one embodiment,greater than 80% of the composition is stable mono-PEG-bindingpolypeptide, more preferably at least 90%, and most preferably at least95%.

In another embodiment, the pegylated polypeptides will preferably retainat least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of thebiological activity associated with the unmodified protein. In oneembodiment, biological activity refers to its ability to bind to atarget molecule, as assessed by K_(D), k_(on) or k_(off). In onespecific embodiment, the pegylated binding polypeptide protein shows anincrease in binding to a target molecule relative to unpegylated bindingpolypeptide.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t½) which is enhancedrelative to the half-life of the unmodified protein. The half-life ofPEG-binding polypeptide may be enhanced by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%or 500%, or even by 1000% relative to the half-life of the unmodifiedbinding polypeptide. In some embodiments, the protein half-life isdetermined in vitro, such as in a buffered saline solution or in serum.In other embodiments, the protein half-life is an in vivo half life,such as the half-life of the protein in the serum or other bodily fluidof an animal.

Deimmunization of Multivalent Polypeptides

In one aspect, the application provides deimmunized multivalentpolypeptides. In some embodiments, the sequence of a multivalentpolypeptide has been altered to eliminate one or more B- or T-cellepitopes.

The multivalent polypeptide may be deimmunized to render itnon-immunogenic, or less immunogenic, to a given species. Deimmunizationcan be achieved through structural alterations to the polypeptide. Anydeimmunization technique known to those skilled in the art can beemployed. One suitable technique, for example, for deimmunizing proteinsis described in WO 00/34317, the disclosure of which is incorporatedherein in its entirety. In summary, a typical protocol within thegeneral method described therein includes the following steps:

1. Determining the amino acid sequence of the polypeptide;

2. Identifying potential T-cell epitopes within the amino acid sequenceof the polypeptide by any method including determination of the bindingof peptides to MHC molecules, determination of the binding of peptide:HLA complexes to the T-cell receptors from the species to receive thetherapeutic protein, testing of the polypeptide or parts thereof usingtransgenic animals with HLA molecules of the species to receive thetherapeutic protein, or testing such transgenic animals reconstitutedwith immune system cells from the species to receive the therapeuticprotein; and3. By genetic engineering or other methods for producing modifiedpolypeptide, altering the polypeptide to remove one or more of thepotential T-cell epitopes and producing such an altered polypeptide fortesting.

In one embodiment, the sequences of the polypeptide can be analyzed forthe presence of MHC class II binding motifs. For example, a comparisonmay be made with databases of MHC-binding motifs such as, for example bysearching the “motifs” database on the worldwide web atsitewehil.wehi.edu.au. Alternatively, MHC class II binding peptides maybe identified using computational threading methods such as thosedevised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)) wherebyconsecutive overlapping peptides from the polypeptide are testing fortheir binding energies to MHC class II proteins. Computational bindingprediction algorithms include iTope™, Tepitope, SYFPEITHI, EpiMatrix(EpiVax), and MHCpred. In order to assist the identification of MHCclass II-binding peptides, associated sequence features which relate tosuccessfully presented peptides such as amphipathicity and Rothbardmotifs, and cleavage sites for cathepsin B and other processing enzymescan be searched for.

Having identified potential (e.g. human) T-cell epitopes, these epitopesare then eliminated by alteration of one or more amino acids, asrequired to eliminate the T-cell epitope. Usually, this will involvealteration of one or more amino acids within the T-cell epitope itself.This could involve altering an amino acid adjacent the epitope in termsof the primary structure of the protein or one which is not adjacent inthe primary structure but is adjacent in the secondary structure of themolecule. The usual alteration contemplated will be amino acidsubstitution, but it is possible that in certain circumstances aminoacid addition or deletion will be appropriate. All alterations can beaccomplished by recombinant DNA technology, so that the final moleculemay be prepared by expression from a recombinant host, for example bywell established methods, but the use of protein chemistry or any othermeans of molecular alteration may also be used.

Once identified T-cell epitopes are removed, the deimmunized sequencemay be analyzed again to ensure that new T-cell epitopes have not beencreated and, if they have, the epitope(s) can be deleted.

Not all T-cell epitopes identified computationally need to be removed. Aperson skilled in the art will appreciate the significance of the“strength” or rather potential immunogenicity of particular epitopes.The various computational methods generate scores for potentialepitopes. A person skilled in the art will recognize that only the highscoring epitopes may need to be removed. A skilled person will alsorecognize that there is a balance between removing potential epitopesand maintaining binding affinity of the polypeptide. Therefore, onestrategy is to sequentially introduce substitutions into the polypeptideand then test for antigen binding and immunogenicity.

In one aspect the deimmunized polypeptide is less immunogenic (orrather, elicits a reduced HAMA response) than the original polypeptidein a human subject. Assays to determine immunogenicity are well withinthe knowledge of the skilled person. Art-recognized methods ofdetermining immune response can be performed to monitor a HAMA responsein a particular subject or during clinical trials. Subjects administereddeimmunized polypeptide can be given an immunogenicity assessment at thebeginning and throughout the administration of said therapy. The HAMAresponse is measured, for example, by detecting antibodies to thedeimmunized polypeptide, in serum samples from the subject using amethod known to one in the art, including surface plasmon resonancetechnology (BIACORE) and/or solid-phase ELISA analysis. Alternatively,in vitro assays designed to measure a T-cell activation event are alsoindicative of immunogenicity.

Additional Modifications

In some embodiments, the multivalent polypeptides are glycosylated. Fn3domains do not normally contain glycosylation sites, however, suchglycosylation may be engineered into the protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.These can be engineered into the proteins of the invention, inparticular fibronectin-based scaffold proteins and their correspondingpolynucleotides. Thus, the presence of either of these tripeptidesequences in a polypeptide creates a potential glycosylation site.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to proteins is conveniently accomplishedby altering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

In some embodiments, the multivalent polypeptides are modified toenhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/orcomplement dependent cytotoxicity (CDC). In some embodiments, themultivalent polypeptide is an Fn3 domain further comprising an Fcregion. In some embodiments, the Fc region is a variant that enhancesADCC or CDC. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g., a substitution) at one or more aminoacid positions.

In one embodiment, the variant Fc region may mediate antibody-dependentcell-mediated cytotoxicity (ADCC) in the presence of human effectorcells more effectively, or bind an Fc gamma receptor (FcγR) with betteraffinity, than a native sequence Fc region. Such Fc region variants maycomprise an amino acid modification at any one or more of positions 256,290, 298, 312, 326, 330, 333, 334, 360, 378 or 430 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

Nucleic Acid-Protein Fusion Technology

In one aspect, the application provides multivalent polypeptidescomprising fibronectin type III domains that bind a human target, suchas, for example, EGFR, VEGFR2, IGF-IR, or other proteins. One way torapidly make and test Fn3 domains with specific binding properties isthe nucleic acid-protein fusion technology of Adnexus, a Bristol-MyersSquibb R&D Company. This disclosure describes the use of such in vitroexpression and tagging technology, termed PROfusion™, that exploitsnucleic acid-protein fusions (RNA- and DNA-protein fusions) to identifynovel polypeptides and amino acid motifs that are important for bindingto proteins. Nucleic acid-protein fusion technology is a technology thatcovalently couples a protein to its encoding genetic information. For adetailed description of the RNA-protein fusion technology andfibronectin-based scaffold protein library screening methods see Szostaket al., U.S. Pat. Nos. 6,258,558; 6,261,804; 6,214,553; 6,281,344;6,207,446; 6,518,018; PCT Publication Nos. WO00/34784; WO01/64942;WO02/032925; and Roberts and Szostak, Proc Natl. Acad. Sci.94:12297-12302, 1997, herein incorporated by reference. Furtherdiscussion of nucleic acid-protein fusion technology can be found in theExamples and the Materials and Methods section of the application.

Vectors & Polynucleotides Embodiments

Nucleic acids encoding any of the various proteins or polypeptidesdisclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc Natl Acad Sci USA. 2003 Jan. 21; 100(2):438-42;Sinclair et al. Protein Expr Purif. 2002 October; 26(1):96-105; ConnellN D. Curr Opin Biotechnol. 2001 October; 12(5):446-9; Makrides et al.Microbiol Rev. 1996 September; 60(3):512-38; and Sharp et al. Yeast.1991 October; 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process a native signal sequence, the signal sequence issubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces alpha-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in PCT Publication No. WO90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor regions may be ligated in readingframe to DNA encoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

A suitable selection gene for use in yeast is the trpl gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrpl gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trpllesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein of the invention, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tac promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein of the invention.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP Patent Publication No. 73,657. Yeast enhancers also areadvantageously used with yeast promoters.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human.beta.-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus.Alternatively, the rous sarcoma virus long terminal repeat can be usedas the promoter.

Transcription of a DNA encoding proteins of the invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, .alpha.-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to themultivalent antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the multivalent antibody.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York,1985), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47,1988). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

Suitable host cells for the expression of glycosylated proteins of theinvention are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

In some instance it will be desired to produce proteins in vertebratecells, such as for glycosylation, and propagation of vertebrate cells inculture (tissue culture) has become a routine procedure. Examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59. (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; ahuman hepatoma line (Hep G2); and myeloma or lymphoma cells (e.g., YO,J558L, P3 and NSO cells) (see U.S. Pat. No. 5,807,715). Plant cellcultures of cotton, corn, potato, soybean, petunia, tomato, and tobaccocan also be utilized as hosts.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.In the examples shown here, the host cells used for high-throughputprotein production (HTPP) and mid-scale production was theHMS174-bacterial strain.

The host cells used to produce the proteins of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO90/03430; WO87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

Proteins of the invention can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The proteins of the present invention can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

Imaging, Diagnostic and Other Applications

In one aspect, the application provides multivalent polypeptides labeledwith a detectable moiety. The polypeptides may be used for a variety ofdiagnostic applications. The detectable moiety can be any one which iscapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas H3, C14 or 13, P32, S35, or I131; a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, rhodamine, or luciferin;or an enzyme, such as alkaline phosphatase, beta-galactosidase orhorseradish peroxidase.

Any method known in the art for conjugating a protein to the detectablemoiety may be employed, including those methods described by Hunter, etal., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974);Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982). In vitro methods, include conjugationchemistry well know in the art including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a moiety (such as PEG) to a protein of theinvention, a linking group or reactive group is used. Suitable linkinggroups are well known in the art and include disulfide groups, thioethergroups, acid labile groups, photolabile groups, peptidase labile groupsand esterase labile groups. Preferred linking groups are disulfidegroups and thioether groups depending on the application. Forpolypeptides without a Cys amino acid, a Cys can be engineered in alocation to allow for activity of the protein to exist while creating alocation for conjugation.

Multivalent binding polypeptides linked with a detectable moiety alsoare useful for in vivo imaging. The polypeptide may be linked to aradio-opaque agent or radioisotope, administered to a subject,preferably into the bloodstream, and the presence and location of thelabeled protein in the subject is assayed. This imaging technique isuseful in the staging and treatment of malignancies. The protein may belabeled with any moiety that is detectable in a subject, whether bynuclear magnetic resonance, radiology, or other detection means known inthe art.

Multivalent polypeptides also are useful as affinity purificationagents. In this process, the polypeptides are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art.

Multivalent polypeptides can be employed in any known assay method, suchas competitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987)).

In certain aspects, the disclosure provides methods for detecting atarget molecule in a sample. A method may comprise contacting the samplewith a multivalent polypeptide described herein, wherein said contactingis carried out under conditions that allow polypeptide-target complexformation; and detecting said complex, thereby detecting said target insaid sample. Detection may be carried out using any technique known inthe art, such as, for example, radiography, immunological assay,fluorescence detection, mass spectroscopy, or surface plasmon resonance.The sample will often by a biological sample, such as a biopsy, andparticularly a biopsy of a tumor, a suspected tumor. The sample may befrom a human or other mammal. The multivalent polypeptide may be labeledwith a labeling moiety, such as a radioactive moiety, a fluorescentmoiety, a chromogenic moiety, a chemiluminescent moiety, or a haptenmoiety. The multivalent polypeptide may be immobilized on a solidsupport.

Therapeutic/In Vivo Uses

In one aspect, the application provides multivalent polypeptides usefulin the treatment of disorders. The application also provides methods foradministering multivalent polypeptides to a subject. In someembodiments, the subject is a human. In some embodiments, themultivalent polypeptides are pharmaceutically acceptable to a mammal, inparticular a human. A “pharmaceutically acceptable” polypeptide refersto a polypeptide that is administered to an animal without significantadverse medical consequences. Examples of pharmaceutically acceptablemultivalent polypeptides include ¹⁰Fn3 domains that lack theintegrin-binding domain (RGD) and ¹⁰Fn3 domains that are essentiallyendotoxin free or have very low endotoxin levels.

The multivalent polypeptides are particularly useful in disorders suchas cancer. In an exemplary embodiment, the multivalent polypeptidecomprises Fn3 domains that bind to two different targets. The twotargets may be within the same signaling pathway or within two separatesignaling pathways. One of the target molecules may “recruit” themultivalent polypeptide to a particular site, such as a cancer cell,while binding to the second target molecule may affect a particularsignaling pathway.

One aspect of the applications provides multivalent polypeptides thattarget one or more proteins involved in tumor biology, such as, forexample, VEGFR2, IGF-IR, and EGFR. In some embodiments, administrationof a multivalent polypeptide inhibits tumor cell growth in vivo. Thetumor cell may be derived from any cell type including, withoutlimitation, epidermal, epithelial, endothelial, leukemia, sarcoma,multiple myeloma, or mesodermal cells. Examples of common tumor celllines for use in xenograft tumor studies include A549 (non-small celllung carcinoma) cells, DU-145 (prostate) cells, MCF-7 (breast) cells,Colo 205 (colon) cells, 3T3/]GF-IR (mouse fibroblast) cells, NCI H441cells, HEP G2 (hepatoma) cells, MDA MB 231 (breast) cells, HT-29 (colon)cells, MDA-MB-435s (breast) cells, U266 cells, SH-SY5Y cells, Sk-Mel-2cells, NCI-H929, RPM18226, and A431 cells. In some embodiments, thepolypeptide inhibits tumor cell growth relative to the growth of thetumor in an untreated animal. In some embodiments, the polypeptideinhibits tumor cell growth by 50, 60, 70, 80% or more relative to thegrowth of the tumor in an untreated animal. In some embodiments, theinhibition of tumor cell growth is measured at least 7 days or at least14 days after the animals have started treatment with the polypeptide.In some embodiments, another antineoplastic agent is administered to theanimal with the polypeptide.

In certain aspects, the disclosure provides methods for administeringmultivalent polypeptides for the treatment and/or prophylaxis of tumoursand/or tumour metastases, where the tumour is particularly preferablyselected from the group consisting of brain tumour, tumour of theurogenital tract, tumour of the lymphatic system, stomach tumour,laryngeal tumour, monocytic leukemia, lung adenocarcinoma, small-celllung carcinoma, pancreatic cancer, glioblastoma and breast carcinoma,without being restricted thereto.

In certain aspects, the disclosure provides methods for administeringmultivalent polypeptide for the treatment of diseases selected from thegroup of cancerous diseases consisting of squamous cell carcinoma,bladder cancer, stomach cancer, liver cancer, kidney cancer, colorectalcancer, breast cancer, head cancer, neck cancer, oesophageal cancer,gynecological cancer, thyroid cancer, lymphoma, chronic leukemia andacute leukemia.

Additional Agents that May be Used with Appropriate Embodiments of theInvention

One aspect of the invention provides multivalent polypeptides linked toa cytotoxic agent. Such embodiments can be prepared by in vitro or invivo methods as appropriate. In vitro methods, include conjugationchemistry well know in the art including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a cytotoxic agent to a polypeptide, a linkinggroup or reactive group is used. Suitable linking groups are well knownin the art and include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups and esterase labilegroups. Preferred linking groups are disulfide groups and thioethergroups. For example, conjugates can be constructed using a disulfideexchange reaction or by forming a thioether bond between the antibodyand the cytotoxic agent. Preferred cytotoxic agents are maytansinoids,taxanes and analogs of CC-1065.

In some embodiments, a multivalent polypeptide is linked to a bacterialtoxin, a plant toxin, ricin, abrin, a ribonuclease (RNase), DNase I, aprotease, Staphylococcal enterotoxin-A, pokeweed antiviral protein,gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin,Ranpimase (Rap), Rap (N69Q), an enzyme, or a fluorescent protein.

In some embodiments, a multivalent polypeptide is linked tomaytansinoids or maytansinoid analogs. Examples of suitablemaytansinoids include maytansinol and maytansinol analogs. Suitablemaytansinoids are disclosed in U.S. Pat. Nos. 4,424,219; 4,256,746;4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650;4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410;5,475,092; 5,585,499; and 5,846,545.

In some embodiments, a multivalent polypeptide is linked to a taxanes.Taxanes suitable for use in the present invention are disclosed in U.S.Pat. Nos. 6,372,738 and 6,340,701.

In some embodiments, a multivalent is linked to CC-1065 or its analogs.CC-1065 and its analogs are disclosed in U.S. Pat. Nos. 6,372,738;6,340,701; 5,846,545 and 5,585,499.

An attractive candidate for the preparation of such cytotoxic conjugatesis CC-1065, which is a potent anti-tumor antibiotic isolated from theculture broth of Streptomyces zelensis. CC-1065 is about 1000-fold morepotent in vitro than are commonly used anti-cancer drugs, such asdoxorubicin, methotrexate and vincristine (B. K. Bhuyan et al., CancerRes., 42, 3532-3537 (1982)).

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, andcalicheamicin are also suitable for the preparation of conjugates of thepresent invention, and the drug molecules can also be linked tomultivalent polypeptides through an intermediary carrier molecule suchas serum albumin.

In other therapeutic treatments or compositions, multivalentpolypeptides are co-administered, or administered sequentially, with oneor more additional therapeutic agents. Suitable therapeutic agentsinclude, but are not limited to, targeted therapeutics, other targetedbiologics, and cytotoxic or cytostatic agents. In some instances in willbe preferred to administer agents from the same or separatetherapeutically acceptable vial, syringe or other administration devicethat holds a liquid formulation.

Cancer therapeutic agents are those agents that seek to kill or limitthe growth of cancer cells while having minimal effects on the patient.Thus, such agents may exploit any difference in cancer cell properties(e.g., metabolism, vascularization or cell-surface antigen presentation)from healthy host cells. Differences in tumor morphology are potentialsites for intervention: for example, the second therapeutic can be anantibody such as an anti-VEGF antibody that is useful in retarding thevascularization of the interior of a solid tumor, thereby slowing itsgrowth rate. Other therapeutic agents include, but are not limited to,adjuncts such as granisetron HCl, androgen inhibitors such as leuprolideacetate, antibiotics such as doxorubicin, antiestrogens such astamoxifen, antimetabolites such as interferon alpha-2a, cytotoxic agentssuch as taxol, enzyme inhibitors such as ras farnesyl-transferaseinhibitor, immunomodulators such as aldesleukin, and nitrogen mustardderivatives such as melphalan HCl, and the like.

The therapeutic agents that can be combined with multivalentpolypeptides for improved anti-cancer efficacy include diverse agentsused in oncology practice (Reference: Cancer, Principles & Practice ofOncology, DeVita, V. T., Hellman, S., Rosenberg, S. A., 6th edition,Lippincott-Raven, Philadelphia, 2001), such as docetaxel, paclitaxel,doxorubicin, epirubicin, cyclophosphamide, trastuzumab, capecitabine,tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane,goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide,bevacizumab, 5-fluorouracil, leucovorin, levamisole, irinotecan,etoposide, topotecan, gemcitabine, vinorelbine, estramustine,mitoxantrone, abarelix, zoledronate, streptozocin, rituximab,idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine,ibritumomab, tositumomab, interferon alpha-2b, melphalam, bortezomib,altretamine, asparaginase, gefitinib, erlonitib, anti-EGF receptorantibody (e.g., cetuximab or panitumab), ixabepilone, epothilones orderivatives thereof, and conjugates of cytotoxic drugs and antibodiesagainst cell-surface receptors. Preferred therapeutic agents areplatinum agents (such as carboplatin, oxaliplatin, cisplatin), taxanes(such as paclitaxel, docetaxel), gemcitabine, and camptothecin.

The one or more additional therapeutic agents can be administeredbefore, concurrently, or after the multivalent polypeptides. The skilledartisan will understand that for each therapeutic agent there may beadvantages to a particular order of administration. Similarly, theskilled artisan will understand that for each therapeutic agent, thelength of time between which the agent, and an antibody, antibodyfragment or conjugate of the invention is administered, will vary.

Formulation and Administration

The application further provides pharmaceutically acceptablecompositions comprising the multivalent polypeptides described herein,wherein the composition is essentially endotoxin free. Therapeuticformulations comprising multivalent polypeptides are prepared forstorage by mixing the described proteins having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Examples of combinations of active compounds are provided inherein. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the proteins of the invention, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated proteins of the invention may remain in thebody for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

While the skilled artisan will understand that the dosage of eachtherapeutic agent will be dependent on the identity of the agent, thepreferred dosages can range from about 10 mg/square meter to about 2000mg/square meter, more preferably from about 50 mg/square meter to about1000 mg/square meter.

For therapeutic applications, the multivalent polypeptides areadministered to a subject, in a pharmaceutically acceptable dosage form.They can be administered intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. The protein may also be administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. Suitable pharmaceuticallyacceptable carriers, diluents, and excipients are well known and can bedetermined by those of skill in the art as the clinical situationwarrants. Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, pH about 7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The method of thepresent invention can be practiced in vitro, in vivo, or ex vivo.

Administration of multivalent polypeptides, and one or more additionaltherapeutic agents, whether co-administered or administeredsequentially, may occur as described above for therapeutic applications.Suitable pharmaceutically acceptable carriers, diluents, and excipientsfor co-administration will be understood by the skilled artisan todepend on the identity of the particular therapeutic agent beingco-administered.

When present in an aqueous dosage form, rather than being lyophilized,the protein typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted. For the treatment of disease, the appropriate dosage ofmultivalent polypeptides will depend on the type of disease to betreated, as defined above, the severity and course of the disease,whether the antibodies are administered for preventive or therapeuticpurposes, the course of previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The protein is suitably administered to the patient at onetime or over a series of treatments.

ADDITIONAL PATENT REFERENCES

Methods and compositions described in the following additional patentApplications and patents are also included in this disclosure:

-   U.S. Publication Nos. 20050186203; 20050084906; 20050008642;    20040202655; 20040132028; 20030211078; 20060083683; 20060099205;    20060228355; 20040081648; 20040081647; 20050074865; 20040259155;    20050038229; 20050255548; 20060246059; and U.S. Pat. Nos. 5,707,632;    6,818,418; and 7,115,396; and PCT International Application    Publication Nos. WO2005/085430; WO2004/019878; WO2004/029224;    WO2005/056764; WO2001/064942; and WO2002/032925.

INCORPORATION BY REFERENCE

All documents and references, including patent documents and websites,described herein are individually incorporated by reference to into thisdocument to the same extent as if there were written in this document infull or in part.

SEQUENCE LISTING ¹⁰Fn3 with the BC, DE, and FG loops underlined(SEQ ID NO: 1) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT 385A08 BC loop(SEQ ID NO: 2) SWSARLKVAR 385A08 DE loop (SEQ ID NO: 3) PKNVYT385A08 FG loop (SEQ ID NO: 4) TRFRDYQP V2B BC loop (SEQ ID NO: 5)SWRHPHFPTR V2B DE loop (SEQ ID NO: 6) PLQPPT V2B FG loop (SEQ ID NO: 7)TDGRNGRLLSIP 385A08-Fn-V2B (Ser tail) (SEQ ID NO: 8)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSTSTSTVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYR TEIDKPSQ385A08-Fn-V2B (Cys tail) (SEQ ID NO: 9)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSTSTSTVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYR TEIDKPCQ385A08-GS5 (SEQ ID NO: 21)-V2B (Ser tail) (SEQ ID NO: 10)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISI NYRTEIDKPSQ385A08-GS10 (SEQ ID NO: 22)-V2B (Ser tail) (SEQ ID NO: 11)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRN GRLLSIPISINYRTEIDKPSQV2B-Fn-385A08 (Ser tail) (SEQ ID NO: 12)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSTSTSTVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQ (SEQ ID NO: 2) V2B-Fn-385A08 (Cys tail) (SEQ ID NO: 13)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSTSTSTVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYR TEIDKPCQV2B-GS5 (SEQ ID NO: 21)-385A08 (Ser tail) (SEQ ID NO: 14)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISI NYRTEIDKPSQV2B-GS10 (SEQ ID NO: 22)-385A08 (Ser tail) (SEQ ID NO: 15)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVT RFRDYQPISINYRTEIDKPSQV2B (Ser tail) (SEQ ID NO: 16)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEI DKPSQ Ser tail(SEQ ID NO: 17) EIDKPSQ Cys tail (SEQ ID NO: 18) EIDKPCQ Short tail(SEQ ID NO: 19) EIDK Fn based linker (SEQ ID NO: 20) PSTSTST GS₅ linker(SEQ ID NO: 21) GSGSGSGSGS GS₁₀ linker (SEQ ID NO: 22)GSGSGSGSGSGSGSGSGSGS (GGGGS)₃ (SEQ ID NO: 23) GGGGS GGGGS GGGGS (GGGGS)₅(SEQ ID NO: 24) GGGGS GGGGS GGGGS GGGGS GGGGS G₄SG₄SG₃SG (SEQ ID NO: 25)GGGGSGGGGSGGGSG AT577 (SEQ ID NO: 26)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPS Q AT580(SEQ ID NO: 27) MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPC Q V2B short(SEQ ID NO: 28) GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPCQ385A08-GPG (SEQ ID NO: 32)-V2B (SEQ ID NO: 29)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTGPGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT385A08-GPGPGPG (SEQ ID NO: 33)-V2B (SEQ ID NO: 30)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTGPGPGPGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT385A08-GPGPGPGPGPG (SEQ ID NO: 34)-V2B (SEQ ID NO: 31)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTGPGPGPGPGPGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYR T (SEQ ID NO: 32) GPG(SEQ ID NO: 33) GPGPGPG (SEQ ID NO: 34) GPGPGPGPGPG 385A08 Core(SEQ ID NO: 35) EVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRT385A08 Core with Full N-terminal Extension (underlined) (SEQ ID NO: 36)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATIS GLKPGVDYTITVYAVTRFRDYQPISINYRT385A08 Core with Full N-terminal Extension(underlined) and Short Tail (underlined) (SEQ ID NO: 37)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDK385A08 Core with N-terminal Extension(underlined) and Ser Tail (underlined) (SEQ ID NO: 38)VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQ385A08 Core with N-terminal Extension(underlined) and Cys Tail (underlined) (SEQ ID NO: 39)VSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPCQ V2B Core(SEQ ID NO: 40) EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTV2B Core with N-terminal Extension (underlined) (SEQ ID NO: 41)VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTV2B Core with full N-terminal Extension(underlined) and Short Tail (underlined) (SEQ ID NO: 42)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEI DKV2B Core with full N-terminal Extension (underlined) and Ser Tail (underlined) (SEQ ID NO: 43)VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDK PSQV2B Core with full N-terminal Extension(underlined) and Cys Tail (underlined) (SEQ ID NO: 44)VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDK PCQFull N-Terminal Extension (SEQ ID NO: 45) MGVSDVPRDLN-Terminal Extension (SEQ ID NO: 46) VSDVPRDL N-Terminal Extension(SEQ ID NO: 48) GVSDVPRDL PA3 linker (SEQ ID NO: 60) PAPAPA PA6 linker(SEQ ID NO: 61) PAPAPAPAPAPA PA9 linker (SEQ ID NO: 62)PAPAPAPAPAPAPAPAPA 385A08-Fn-V2B (Modified Ser tail) (SEQ ID NO: 63)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPSTSTSTVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEG SGS385A08-Fn-V2B (Modified Cys tail) (SEQ ID NO: 64)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPSTSTSTVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEG SGC385A08-PA3 (SEQ ID NO: 60)-V2B (Modified Ser tail) (SEQ ID NO: 65)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPAPAPAVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEGS GS385A08-PA3 (SEQ ID NO: 60)-V2B (Modified Cys tail) (SEQ ID NO: 66)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPAPAPAVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEGS GC385A08-PA6 (SEQ ID NO: 61)-V2B (Modified Ser tail) (SEQ ID NO: 67)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPAPAPAPAPAPAVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISIN YRTEGSGS385A08-PA6 (SEQ ID NO: 61)-V2B (Modified Cys  tail) (SEQ ID NO: 68)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPAPAPAPAPAPAVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISIN YRTEGSGC385A08-PA9 (SEQ ID NO: 62)-V2B (Modified Ser tail) (SEQ ID NO: 69)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPAPAPAPAPAPAPAPAPAVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLS IPISINYRTEGSGS385A08-PA9 (SEQ ID NO: 62)-V2B (Modified Cys  tail) (SEQ ID NO: 70)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEPAPAPAPAPAPAPAPAPAVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLS IPISINYRTEGSGCModified Ser tail (SEQ ID NO: 71) EGSGS Modified Cys tail(SEQ ID NO: 72) EGSGC

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

Example 1. Multivalent Fibronectin Scaffold Domain Proteins, IncludingSeveral V/I ¹⁰Fn3-Based Binders

Various constructs of multivalent fibronectin scaffold domain proteinswere generated. The following table depicts constructs described hereinand their SEQ ID NOS.

Construct Description SEQ ID NO: 385A08-Fn-V2B A bivalent I/V constructhaving the 8 structure (SEQ ID NO: 37)-(SEQ ID NO: 20)-(SEQ ID NO: 43)385A08-Fn-V2B-Cys A bivalent I/V construct having the 9 structure (SEQID NO: 37)-(SEQ ID NO: 20)-(SEQ ID NO: 44) 385A08-GS5 (SEQ ID A bivalentI/V construct having the 10 NO: 21)-V2B structure (SEQ ID NO: 37)-(SEQID NO: 21)-(SEQ ID NO: 43) 385A08-GS10 (SEQ ID A bivalent I/V constructhaving the 11 NO: 22)-V2B structure (SEQ ID NO: 37)-(SEQ ID NO: 22)-(SEQID NO: 43) V2B-Fn-385A08 A bivalent V/I construct having the 12structure (SEQ ID NO: 42)-(SEQ ID NO: 20)-(SEQ ID NO: 38)V2B-Fn-385A08-Cys A bivalent V/I construct having the 13 structure (SEQID NO: 42)-(SEQ ID NO: 20)-(SEQ ID NO: 39) V2B-GS5 (SEQ ID A bivalentV/I construct having the 14 NO: 21)-385A08 structure (SEQ ID NO:42)-(SEQ ID NO: 21)-(SEQ ID NO: 38) V2B-GS10 (SEQ ID A bivalent V/Iconstruct having the 15 NO: 22)-385A08 structure (SEQ ID NO: 42)-(SEQ IDNO: 22)-(SEQ ID NO: 38) 385A08-GPG (SEQ ID A bivalent I/V constructhaving the 29 NO: 32)-V2B structure (SEQ ID NO: 36)-(SEQ ID NO: 32)-(SEQID NO: 41) 385A08-GPGPGPG A bivalent I/V construct having the 30 (SEQ IDNO: 33)-V2B structure (SEQ ID NO: 36)-(SEQ ID NO: 33)-(SEQ ID NO: 41)385A08- A bivalent I/V construct having the 31 GPGPGPGPGPG (SEQstructure (SEQ ID NO: 36)-(SEQ ID NO: ID NO: 34)-V2B 34)-(SEQ ID NO: 41)V2BShort A VEGFR2 binder having the V2B Core 28 with a Gly at theN-terminus and the Cys tail; the structure is Gly-(SEQ ID NO: 40)- (SEQID NO: 18) Peg-V2Bshort V2B Short which is pegylated at position 93 28V2B (Ser tail) A VEGFR2 binder having the V2B Core 16 with a fullN-terminal extension and a Ser tail; the structure is (SEQ ID NO:45)-(SEQ ID NO: 40)-(SEQ ID NO: 17) AT577 An IGF-IR binder having the385A08 Core 26 with a full N-terminal extension and a Ser tail; thestructure is (SEQ ID NO: 45)-(SEQ ID NO: 35)-(SEQ ID NO: 17) AT580 AnIGF-IR binder having the 385A08 Core 27 with a full N-terminal extensionand a Cys tail; the structure is (SEQ ID NO: 45)-(SEQ ID NO: 35)-(SEQ IDNO: 18) AT580-PEG40 AT580 which has been pegylated with a 40 27 kDa PEGat position 98 AT580-PEG20-AT580 A bivalent I/I construct which has two27 AT580 molecules attached via a 20 kDa PEG molecule at position 98 ofeach molecule 385A08 Core 385A08 is an exemplary IGF-IR binder 35 AnIGF-IR binder having the 385A08 Core 36 with Full N-terminal Extension;the structure is (SEQ ID NO: 35)-(SEQ ID NO: 45) An IGFR-IR binderhaving the 385A08 37 Core with Full N-terminal Extension and Short Tail;the structure is (SEQ ID NO: 45)-(SEQ ID NO: 35)-(SEQ ID NO: 19) AnIGFR-IR binder having the 385A08 38 Core with N-terminal Extension andSer Tail; the structure is (SEQ ID NO: 46)- (SEQ ID NO: 35)-(SEQ ID NO:17) An IGFR-IR binder having the 385A08 39 Core with N-terminalExtension and Ser Tail; the structure is (SEQ ID NO: 46)- (SEQ ID NO:35)-(SEQ ID NO: 18) V2B Core V2B is an exemplary VEGFR2 binder 40 AVEGFR2 binder having the V2B Core 41 with N-terminal Extension; thestructure is (SEQ ID NO: 46)-(SEQ ID NO: 40) A VEGFR2 binder having theV2B Core 42 with Full N-terminal Extension and Short Tail; the structureis (SEQ ID NO: 45)- (SEQ ID NO: 40)-(SEQ ID NO: 19) A VEGFR2 binderhaving the V2B Core 43 with Full N-terminal Extension and Ser Tail; thestructure is (SEQ ID NO: 45)- (SEQ ID NO: 40)-(SEQ ID NO: 17) A VEGFR2binder having the V2B Core 44 with Full N-terminal Extension and CysTail; the structure is (SEQ ID NO: 45)- (SEQ ID NO: 40)-(SEQ ID NO: 18)385A08-Fn-V2B A bivalent I/V construct having the 63 (Modified Ser tail)structure (SEQ ID NO: 36)-E-(SEQ ID NO: 20)-(SEQ ID NO: 41)-(SEQ ID NO:71) 385A08-Fn-V2B A bivalent I/V construct having the 64 (Modified Cystail) structure (SEQ ID NO: 36)-E-(SEQ ID NO: 20)-(SEQ ID NO: 41)-(SEQID NO: 72) 385A08-PA3 (SEQ ID A bivalent I/V construct having the 65 NO:60)-V2B structure (SEQ ID NO: 36)-E-(SEQ ID NO: (Modified Ser tail)60)-(SEQ ID NO: 41)-(SEQ ID NO: 71) 385A08-PA3 (SEQ ID A bivalent I/Vconstruct having the 66 NO: 60)-V2B structure (SEQ ID NO: 36)-E-(SEQ IDNO: (Modified Cys tail) 60)-(SEQ ID NO: 41)-(SEQ ID NO: 72) 385A08-PA6(SEQ ID A bivalent I/V construct having the 67 NO: 61)-V2B structure(SEQ ID NO: 36)-E-(SEQ ID NO: (Modified Ser tail) 61)-(SEQ ID NO:41)-(SEQ ID NO: 71) 385A08-PA6 (SEQ ID A bivalent I/V construct havingthe 68 NO: 61)-V2B structure (SEQ ID NO: 36)-E-(SEQ ID NO: (Modified Cystail) 61)-(SEQ ID NO: 41)-(SEQ ID NO: 72) 385A08-PA9 (SEQ ID A bivalentI/V construct having the 69 NO: 62)-V2B structure (SEQ ID NO: 36)-E-(SEQID NO: (Modified Ser tail) 62)-(SEQ ID NO: 41)-(SEQ ID NO: 71)385A08-PA9 (SEQ ID A bivalent I/V construct having the 70 NO: 62)-V2Bstructure (SEQ ID NO: 36)-E-(SEQ ID NO: (Modified Cys tail) 62)-(SEQ IDNO: 41)-(SEQ ID NO: 72)

385A08 is an exemplary IGF-IR binder. V2B is an exemplary VEGFR2 binder.The two domains are linked by either glycine-serine based linkers (e.g.,GS5, SEQ ID NO: 21, and GS10, SEQ ID NO: 22) or a modified stretch ofamino acids that connect the first and second human Fn3 domains (SEQ IDNO: 20). These V/I ¹⁰Fn3-based binders can be oriented in differentways—for instance, the V2B domain at the N-terminus, or the IGF-IRdomain at the N-terminus. Peg-V2Bshort is a construct lacking the firsteight amino acids of the fibronectin based scaffold domain of V2B and ispegylated at its single cysteine residue. AT577 is an IGF-IR binder witha C-terminal tail of SEQ ID NO: 17. AT580 is an IGF-IR binder with aC-terminal tail of SEQ ID NO: 18. Specifically, AT580 is represented bySEQ ID NO: 27. AT580-Peg20-AT580 are two IGF-IR binders connected viatheir single cysteine residues with a PEG linkage. AT580-PEG40 is anIGF-IR binder (SEQ ID NO: 27) that has a 40 kD peg attached. Themultivalent constructs used in the following experiments generallycontain a C-terminal His6 tag (SEQ ID NO: 59).

Example 2: Expression of V/I ¹⁰Fn3-Based Binders

Certain V/I ¹⁰Fn3-based binders were purified using a high throughputprotein production process (HTPP) as described in the materials andmethods section below. FIG. 1 depicts an exemplary SDS-PAGE analysisfrom several of the V/I ¹⁰Fn3-based binders, including those linked byan Fn linker, GS5 linker (SEQ ID NO: 21), and GS10 linker (SEQ ID NO:22). FIG. 1 demonstrates acceptable expression levels for the multipleconstructs that were tested. Additionally, select clones were alsopurified using a mid-scale purification process, which encompassed thepurification of insoluble binders. An alternative method can also beused, which encompasses purification of soluble binders. These mid-scalepurification processes are described in the materials and methodssection below. Some specific clones were also purified using a largescale purification process, which is described in the materials andmethods section below.

Example 3: Biophysical Characterization of V/I ¹⁰Fn3-Based Binders

Standard size exclusion chromatography (SEC) was performed on certainHTPP, midscale and large scale purified V/I ¹⁰Fn3-based binders. Massspectrometry (LC-MS) and differential scanning calorimetry (DSC)analyses were also performed on certain of these constructs that werepurified from midscale and large scale processes. Each of theseanalytical methods are described in the materials and methods sectionbelow.

SEC results of the HTPP purified constructs varied depending on theorientation of the 385A08 (IGF-IR domain) or V2B (VEGFR-2 domain)subunits within the tandem. With the 385A08 subunit on the N-terminalside (i.e., 385A08-Fn-V2B with a his-tag), SEC showed predominantlymonomeric protein (eluted in the 23 kDa range vs. globular molecularweight standards), as shown in FIG. 2. On the other hand, when the V2Bsubunit is on the N-terminal side (i.e., V2B-Fn-385A08 with a his-tag,SEQ ID NO: 8), this resulted in a mixture of monomer (23 kDa) and dimer(46 kDa vs. globular molecular weight standards), as shown in FIG. 3.SEC analysis of constructs where a GS5 (SEQ ID NO: 21) or GS10 linker(SEQ ID NO: 22) was used instead of an Fn linker showed similar results,depending on the orientation of the 385A08 and V2B subunits.

SECs of midscale purified constructs 385A08-Fn-V2B (SEQ ID NO: 8) and385A08-Fn-V2B-cys (SEQ ID NO: 9) were also evaluated. For the midscalepurified construct 385A08-Fn-V2B (which has a his-tag, and includes anaturally occurring serine at the C-terminus), the SEC profile waspredominantly monomeric with elution in the approximate range of 23 kDabased on globular molecular weight standards. However, for the cysteineversion of this construct (385A08-Fn-V2B-cys with a his-tag), the SECdemonstrated a mixture of monomeric and dimeric protein. It should benoted that the cysteine version is typically created for pegylationpurposes. Equivalent results were observed for solubly expressed andpurified 385A08-Fn-VB2 (with his tag) and material that was expressed inan insoluble form and refolded.

Select midscale constructs were further analyzed by LC-MS. The molecularweight measured by LC-MS for 385A08-Fn-V2B (with his tag) is 23,260Daltons, which matches the molecular weight of the “desMet form” (aversion of the tandem where the first methionine is cleaved off)calculated from amino acid composition of the tandem. The molecularweight measured by LC-MS for 385A08-Fn-V2B-Cys (with a his-tag) is23,581 Daltons, which is +174 Daltons of the theoretical calculatedmolecular weight based on the amino acid composition of the tandemindicating a post translational modification most likely of the Cysresidue. In addition, the LC-MS for 385A08-GS10 (SEQ ID NO: 22)-V2B(with his-tag, SEQ ID NO: 11) is 24,040, matching the theoreticalmolecular weight for this construct. See FIG. 4.

In addition, Differential Scanning Calorimetry (DSC) analysis wasperformed on two of the midscale purified constructs. The experimentallydetermined T_(m) of 385A08-Fn-V2B (with his tag) in 50 mM NaOAc, 150 mMNaCl, pH 4.5 is equal to 51.49° C. The DSC of 385A08-Fn-V2B-Cys (withhis tag) in 50 mM NaOAc, 150 mM NaCl, pH 4.5 showed two transitionstates of 46.77° C. & 55.52° C. which is probably due to the presence ofdimer due to disulfide bond formation between two tandems (See FIG. 4).

SEC was also performed on large-scale purified, pegylated385A08-Fn-V2B-cys (no his tag, SEQ ID NO: 9). SEC of this constructdemonstrated material that was 95.1% monomeric as shown in FIG. 5. Inaddition, DSC analysis on this same construct demonstrated that it washalf unfolded at a temperature of 56° C. (see FIG. 6) with no unfoldingdetected at up to 45° C.

Example 4: Effects of Pegylation on Multivalent Fibronectin BasedProteins

The fibronectin based scaffold proteins can be pegylated according tothe method described below. Various types of PEG, such as linear orbranched PEGs, and various sized PEGs can be used. In particular,midscale and large scale purified versions of the construct385A08-Fn-V2B-Cys (SEQ ID NO: 9) were pegylated with 40 kD linear and 40kD branched PEG.

An analysis of drug concentration over a 120 hour period in micerevealed that his-tagged 385A08-Fn-V2B-Cys with branched PEGdemonstrated a half life of 14.6 hours, while the linear pegylatedversion of this construct (with his tag) demonstrated a half life of10.5 hours (FIG. 7). Details of the dosing schedule and half lifecalculations are described in the materials and methods section below.All concentrations of Adnectin reported here and in the rest of thedocument are based on the protein and not the PEG portion of thecompound.

Example 5. Determination of Binding Affinity Using Surface PlasmonResonance (BIAcore) Analysis on Multivalent Fibronectin Based Proteins

Selected HTPP purified fibronectin based scaffold proteins, includingseveral V/I ¹⁰Fn3-based binders, were evaluated for their kineticbehavior towards IGF-IR using surface plasmon resonance as described inthe materials and methods section below. The dissociation constant,K_(D), for these clones is shown in the first column of FIG. 8.

A concentration series of the midscale purification of the tandemconstruct 385A08-Fn-V2B (non pegylated, his-tagged version, SEQ ID NO:8) was also evaluated. The functionality of each of the domains of thisconstruct, specifically, the V2B domain, and the IGF-IR domain, wasevaluated as described in the materials and methods section below. Withrespect to the V2B domain, the K_(D) of 385A08-Fn-V2B for VEGFR2-Fc was˜0.3 nM with an average on rate of 1.4×10⁶ M⁻¹ sec-1, the average offrate was 4.5×10⁴ sec⁻¹. With respect to the IGF-IR domain, the K_(D) of385A08-Fn-V2B for IGF-IR-Fc was ˜20 pM with an on rate of 1.1×10⁷ M⁻¹sec⁻¹ and an off rate of 1.7×10⁴ sec⁻¹ (n of 1).

Data was also collected for midscale purified material of the tandem385A08-GS10 (SEQ ID NO: 22)-V2B (SEQ ID NO: 11) and V2B-GS10 (SEQ ID NO:22)-385A08 (SEQ ID NO: 15). Both constructs included a his-tag. TheK_(D) for the IGF-IR domain was 40 pM and 50 pM, respectively. The K_(D)for the V2B domain was 0.5 nM and 0.6 nM, respectively. Additional datawas collected for a linear pegylated, his-tagged midscale purifiedversion of construct 385A08-Fn-V2B-cys (SEQ ID NO: 9). The K_(D) for theIGF-IR domain was 1.2 nM. The K_(D) for the V2B domain was 14 nM. Thesedata are shown in FIG. 9.

Affinity data was also measured from midscale and large scale material,for the pegylated version of the tandem construct 385A08-Fn-V2B-cys (SEQID NO: 9, with and without his tag). The specific materials and methodsfor these calculations are described below. The results show that thevarious constructs tested bound to IGF1R-Fc with an average on-rate of3.15 E+05 M⁻¹ s⁻¹+/−0.94 E+05 M⁻¹ s⁻¹, and an average off-rate of 2.47E−04 s⁻+/−0.18 E−04 s⁻. For VEGFR2-Fc, the measured average associationrate was 1.15 E+04 M⁻¹ s⁻+/−0.62 E+04 M⁻¹ s⁻¹, and the dissociation ratewas 1.02 E−04 s⁻+/−0.21 E−04 s⁻¹. These data are shown in FIG. 10. Theaverage calculated affinities for VEGFR2 and IGF1R using thisexperimental format are 10.5±0.48 nM and 0.84±0.22 nM, respectively.

Example 6: Competitive IGF1R and Competitive VEGFR-2 Blocking Assays

Certain tandem constructs were evaluated for their ability to competewith mono-specific IGF-IR and VEGFR-2 binders in vitro. In particular, apegylated, his-tagged version of the construct 385A08-Fn-V2B-cys (SEQ IDNO: 9) was shown to disrupt the interaction of a mono-specific IGF-IR¹⁰Fn3-based binder (AT580-PEG40, SEQ ID NO: 27) with cell surfaceIGF-IR. Using the cell line R⁺, which overexpresses IGF-IR, we haveshown that the IC₅₀ for blocking the interaction of a high affinitybivalent ¹⁰Fn3-based IGF1R binder (AT580-PEG20-AT580, SEQ ID NO: 27) was900 nM for pegylated, his-tagged 385A08-Fn-V2B-cys, whereas it was 600nM for AT580-PEG40. This suggests that pegylated, his-tagged385A08-Fn-V2B-cys has IGF-IR affinity similar to that of AT580-PEG40.FIG. 11 demonstrates this data.

Similarly, the pegylated, his-tagged construct 385A08-Fn-V2B-cys wasable to block the interaction of a mono-specific ¹⁰Fn3-based VEGFR-2binder (Peg-V2Bshort, SEQ ID NO: 28) with VEGFR-2 present on 293:KDRcells. Results from the 293:KDR assay demonstrated an IC₅₀ ofapproximately 140 nM for the pegylated, his-tagged construct385A08-Fn-V2B-cys, whereas that of Peg-V2Bshort is approximately 20 nM(see FIG. 12). The 7-fold difference in activity between Peg-V2Bshortand the pegylated, his-tagged construct 385A08-Fn-V2B-cys is consistentwith the fact that the VEGFR-2 binding portion of 385A08-Fn-V2B-cys hasapproximately a 5-fold lower affinity for VEGFR-2 as compared toPeg-V2Bshort. In particular, in a Ba/F3 assay (described below), theIC₅₀ for the monomeric, non-pegylated VEGFR2 binder V2Bshort (SEQ ID NO:28) is approximately 0.1-3 nM whereas the IC₅₀ for the monomeric,non-pegylated VEGFR2 binder having SEQ ID NO: 44 (e.g., with anN-terminal extension) is approximately 8-13 nM. Therefore, the versionof the VEGFR2 binder included in the tandem construct (e.g., having SEQID NO: 44 which includes an N-terminal extension) has an approximately5-fold lower affinity for VEGFR2 as compared to the monomeric VEGFR2binder used in this assay (e.g., having SEQ ID NO: 28). Both the R⁺ and293:KDR cell based competitive blocking assays are described in detailin the materials and methods section below.

Example 7. In Vitro Proliferation Assays

Certain purified constructs, including several V/I ¹⁰Fn3-based binders,were evaluated in Ba/F3 and Rh41 cell-based assays in order to confirmactivity. IC50 s are depicted in FIG. 8 (columns 2, 3). In addition,certain purified constructs were evaluated in Rh41 and HMVEC-Lproliferation assays (also described in the materials and methodssection below). These constructs were compared to a mono-specificantibody directed to IGF1R (MAB391), a mono-specific antibody to VEGF(bevacizumab), another mono-specific ¹⁰Fn3-based IGF1R binder (AT-577,SEQ ID NO: 26), a mono-specific ¹⁰Fn3-based VEGFR-2 binder(Peg-V2Bshort, SEQ ID NO: 28), and a wild type ¹⁰Fn3-based protein thatdoes not bind to a target (SGE). The data demonstrates that neither theorientation of the tandem (e.g., whether the I portion or the V portionis at the N-terminus) or the choice of linker affected the results. Thisdata is summarized in FIG. 13. In addition, Ba/F3 and NCI-H929 cellproliferation assays were performed to compare certain his-tag andnon-his-tag versions of the pegylated construct 385A08-Fn-V2B-Cys (SEQID NO: 9). These constructs were compared to MAB391, mono-specific¹⁰Fn3-based IGF-IR binder AT580-PEG40 (SEQ ID NO: 27), and mono-specific¹⁰Fn3-based VEGFR2 binder Peg-V2Bshort (SEQ ID NO: 28). These data aresummarized in FIG. 14.

Example 8. Activation and Signaling Activity of Multivalent FibronectinBased Proteins in Cell-Based Assays

Select V/I ¹⁰Fn3-based binders were screened for the ability to directlyinterfere with ligand-stimulated VEGFR2 and IGF-IR activation anddownstream MAP kinase signaling. HEK/293 cells and Porcine aorticendothelial (PAE) cells were transfected with VEGFR2. Stimulation byVEGF induces phosphorylation of VEGFR and downstream signaling in bothcell types. Stimulation by IGF1 induces phosphorylation of IGF-IR anddownstream signaling in both cell types, see FIG. 15. Further details onthe PAE and HEK/293 assays are discussed in the materials and methodssection below.

FIG. 16 depicts the effect of various multivalent proteins on HEK/293cells. All of the multivalent proteins comprising an IGF-IR bindingfibronectin scaffold domain decreased levels of phosphorylated IGF-IR tosimilar levels as compared to a single IGF-IR binding fibronectinscaffold domain protein (AT577). All of the multivalent proteinscomprising a VEGFR2 binding fibronectin scaffold domain decreased levelsof phosphorylated VEGFR to similar levels as compared to a single VEGFR2binding fibronectin scaffold domain protein (Peg-V2Bshort). Cellproliferation was evaluated by [³H]-thymidine incorporation afterexposure to the various constructs.

FIG. 17 depicts the effect of various multivalent proteins on PAE cells.All of the multivalent proteins comprising an IGF-IR binding fibronectinscaffold domain decreased levels of phosphorylated IGF-IR to similarlevels as compared to a single IGF-IR binding fibronectin scaffolddomain protein (AT577). All of the multivalent proteins comprising aVEGFR2 binding fibronectin scaffold domain decreased levels ofphosphorylated VEGFR to similar levels as compared to a single VEGFR2binding fibronectin scaffold domain protein (Peg-V2Bshort). Cellproliferation was evaluated by [³H]-thymidine incorporation afterexposure to the various constructs.

These results demonstrate that the exemplary polypeptide linkers areable to link fibronectin scaffold domains in an orientation wherebytheir binding to ligand, and therefore their ability to inhibit receptorsignaling, is retained.

Additional studies were conducted on specific varieties of the tandemconstruct 385A08-Fn-V2B-cys (SEQ ID NO: 9, with and without PEG; withand without a his-tag) in primary human microvascular endothelial cellsfrom lung (HMVEC-L). Assays measuring the following were conducted inHMVEC-L: cell proliferation, inhibition of ligand-induced VEGFR-2 andIGF-1R activity (via Western blot analysis), the mobilization ofintracellular calcium (Ca2⁺ Flux) in endothelial cells, and the abilityto inhibit formation of nascent capillary-like structures known astubes. An additional study measuring the inhibition of ligand-inducedVEGFR-2 and IGF-1R activity via Western blot analysis was also conductedin Rh41 cells. Each of these assays are described in further detail inthe materials and methods section below.

The results from the cellular proliferation assay showed that specificvarieties of the 385A08-Fn-V2B-cys constructs inhibit proliferation inHMVEC-L cells with an IC₅₀ ranging between 90 nM and 167 nM (see FIG.18). In one example, a pegylated, non-his-tag version of this constructinhibited proliferation in HMVEC-L cells with an IC₅₀ of ˜167 nM,whereas the mono-specific ¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQID NO: 28) had an IC₅₀ of ˜47 nM.

In order to assess the ability of the 385A08-Fn-V2B-cys constructs toinhibit IGF-1R and VEGFR-2, Western blot analysis was done to measurethe activity of ligand-induced VEGFR-2 and IGF-1R activity in HMVEC-Lcells by autophosphorylation in the presence or absence of the testcompounds. Appropriate controls included the mono-specific ¹⁰Fn3-basedVEGFR-2 binder (Peg-V2Bshort, SEQ ID NO: 28), and the mono-specific¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ ID NO: 27). The constructsgenerally inhibited pVEGFR-2 (pVEGFR-2 is phospho-VEGFR-2; also known asp-Flk-1), pIGF-1R, and pAKT activity (see FIG. 19). A pegylated,non-his-tag version of the 385A08-Fn-V2B-cys construct demonstratedinhibition of pVEGFR-2 activity in a dose response starting at 10 nM,with near complete inhibition being achieved at 100 nM. This samecompound also potently inhibited pIGF-1R activity at between 1 nM and 10nM as compared to AT580-PEG40 where the ICso for inhibition was 100 nM.

In order to assess the ability of the specific varieties of the385A08-Fn-V2B-cys constructs to inhibit IGF-1R in an IGF-1R-driven tumorcell line, Western blot analysis was carried out to measure the activityof ligand-induced IGF-1R activity in Rh41 cells by autophosphorylationin the presence or absence of the test compounds. Inhibition of pIGF-1Ractivity was generally observed for all of the 385A08-Fn-V2B-cysconstructs at the 100 nM dose. Inhibition of pAKT was observed atvarying levels for all of the 385A08-Fn-V2B-cys constructs tested. Inone example, the pegylated, non his-tag version of the 385A08-Fn-V2B-cysconstruct demonstrated inhibition of pIGF-1R activity starting at thedose response in the ˜1-10 nM range and inhibition was nearly completeat 100 nM. These data are summarized in FIG. 20.

Because VEGF stimulates the mobilization of intracellular calcium inendothelial cells (as described in Ku, D. D., et al., Vascularendothelial growth factor induces EDRF-dependent relaxation in coronaryarteries. Am J Physiol, 1993. 265(2 Pt 2): p. H586-92), the ability toinhibit calcium release (Ca²⁺) is another measure of cell-basedsignaling for a VEGFR-2 inhibitor. In an assay measuring calcium releasein HMVEC-L cells, the specific varieties of 385A08-Fn-V2B-cys constructsdemonstrated an inhibition of Ca²⁺ release with an IC₅₀ of 4-10 nM. Asimilar level of inhibition was demonstrated for a mono-specific¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQ ID NO: 28). The results ofthis study are summarized in FIG. 21.

Example 9. Tumor Xenograft Efficacy Studies on Multivalent FibronectinBased Proteins

A pegylated, his-tagged version of the construct 385A08-Fn-V2B-Cys wasevaluated in multiple tumor studies using an RH-41 humanrhabdomyosarcoma model, an A549 human lung model, a GEO colon tumorxenograft model, and an A673 Ewing Sarcoma xenograft tumor model. RH-41and A673 were selected for in vivo testing with 385A08-Fn-V2B-Cys asboth models had previously shown sensitivity to IGF-1R inhibition. A549and GEO, which are not as sensitive to IGF1R inhibition, were selectedto compare the potency of VEGFR2 inhibition by the multivalentconstructs as compared to the monomeric VEGFR2 inhibitor, PEG-V2B-short(SEQ ID NO: 28 with PEG attached at the single cysteine residue). Thesemodels are described in more detail in the materials and methods sectionbelow.

The RH-41 tumor xenograft model has been demonstrated to be sensitive toIGF-IR inhibition. Pegylated, his-tagged 385A08-Fn-V2B-Cys wasadministered on a 3×wk schedule using a single dose level of 100 mg/kg.As illustrated in FIG. 22, segment A, pegylated, his-tagged385A08-Fn-V2B-Cys was effective in inducing tumor growth delay whendosed for three weeks using this regimen. When compared to a combinationof a mono-specific ¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQ ID NO:28) and a mono-specific ¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ IDNO: 27) that should yield a comparable stoichiometry of target bindingfor IGFR-1 and VEGFR-2, pegylated, his-tagged 385A08-Fn-V2B-Cys resultedin similar antitumor activity. Furthermore, the antitumor activityachieved with the multivalent construct was superior to that observedwhen these agents were dosed individually based on percent tumor growthinhibition (% TGI). No overt toxicity was observed for any of theseagents as defined by morbidity, behavioral changes or significant weightloss (e.g., >5%). As summarized in FIG. 22, Segment B, all of theseagents were effective in yielding >50% TGI, however both the combinationof AT580-PEG40, Peg-V2Bshort, as well as pegylated, his-tagged385A08-Fn-V2B-Cys alone, resulted in an increased % TGI when compared toeach agent alone.

The 385A08-Fn-V2B-cys (with Peg) construct binds to human IGF1R with amuch higher affinity than it binds to mouse IGF1R. In particular,385A08-Fn-V2B-cys (with Peg) binds to human IGF1R with a Kd of 277 pM,to monkey IGF1R with a Kd of 213 pM, to rat IGF1R with a Kd of 92,000 pMand to mouse IGF1R with a Kd of 86,000 pM. The tumor xenograft modelsused in this study involve human tumors displaying human IGF1R but alsoinvolve mouse endothelial cells expressing mouse IGF1R. Since thebinding affinity for 385A08-Fn-V2B-cys (with Peg) to mouse IGF1R is somuch lower than that for human IGF1R, the effects seen in this model mayunderestimate the effects of 385A08-Fn-V2B-cys (with Peg) on tumorinhibition. In particular, tumor growth inhibition could be moresignificant if the binding affinity for the IGF1R expressed on theendothelial cells was higher.

In a second study using the RH-41 tumor xenograft model, pegylated,his-tagged 385A08-Fn-V2B-Cys was evaluated over several dose levelsincluding 50 mg/kg, 100 mg/kg and 200 mg/kg. These levels were alsocompared to the combined equivalent API (active pharmaceuticalingredient) dose level of each of the individually targetedmono-specific binders, e.g., a mono-specific ¹⁰Fn3-based VEGFR-2 binder(Peg-V2Bshort, SEQ ID NO: 28) and a mono-specific ¹⁰Fn3-based IGF-IRbinder (AT580-PEG40, SEQ ID NO: 27). The results of this study areillustrated in FIG. 23 where, for the purpose of clarity, each doselevel of pegylated, his-tagged 385A08-Fn-V2B-Cys is shown individuallyin comparison to the corresponding combination dose levels of theindividual AT580-PEG40 and Peg-V2Bshort. The combination of AT580-PEG40and Peg-V2Bshort compared favorably with pegylated, his-tagged385A08-Fn-V2B-Cys, resulting in similar % TGI. Furthermore, all doselevels of pegylated, his-tagged 385A08-Fn-V2B-Cys resulted in similarantitumor activity (as illustrated in FIG. 23, Segment D), ranging from75-83% TGI. As a result, further titration at lower dose levels ofpegylated, his-tagged 385A08-Fn-V2B-Cys will be required in order togenerate a definitive antitumor dose response curve and to determine theminimum efficacious dose level in this tumor model.

The antitumor activity of pegylated, his-tagged 385A08-Fn-V2B-Cys wasalso evaluated in the A549 tumor model in comparison to a mono-specific¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQ ID NO: 28) and amono-specific ¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ ID NO: 27), aswell as a combination treatment with these two mono-specific binders(see FIG. 24). Given the lack of an antitumor response to AT580-PEG40 atthe two dose levels tested (FIG. 26, Segments A & C) and based on lessthan 50% TGI (FIG. 26, Segments B & D), this model appears to beinsensitive to inhibition of IGF-1R. Further, the combination ofAT580-PEG40 and Peg-V2Bshort, did not result in enhanced antitumoractivity when compared to dosing of Peg-V2Bshort alone, which was activeat both dose levels tested. The observation that the level of tumorgrowth inhibition achieved with pegylated, his-tagged 385A08-Fn-V2B-Cyswas comparable to that seen with Peg-V2Bshort dosed alone suggests thatthe antitumor activity observed in this model appears to reside with theantiVEGFR-2 activity of pegylated, his-tagged 385A08-Fn-V2B-Cys andPeg-V2Bshort.

Comparable results were also obtained when these agents were evaluatedin the GEO human colon xenograft model, which also appears to beinsensitive to inhibition of IGF-1R (FIG. 25). In this model, pegylated,his-tagged 385A08-Fn-V2B-Cys was inactive at 100 mg/kg (FIG. 27,Segments A and B) and the mono-specific ¹⁰Fn3-based IGF-IR binder(AT580-PEG40, SEQ ID NO: 27) was inactive at both dose levels tested(FIG. 25, Segments A, B, C, & D). At the higher dose level of 200 mg/kg,pegylated, his-tagged 385A08-Fn-V2B-Cys was active and demonstratedantitumor activity based on >50% TGI (FIG. 25, Segments C and D).Similar to the observations made using the A549 model, the mono-specific¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQ ID NO: 28) was activeagainst the GEO model, suggesting that the antitumor activity observedwith pegylated, his-tagged 385A08-Fn-V2B-Cys in this model may be solelydependent on the anti-VEGFR-2 component and that the GEO model isinsensitive to IGF-IR inhibition. The increased antitumor activityobserved with Peg-V2Bshort (as compared to pegylated, his-tagged385A08-Fn-V2B-Cys) in this model (illustrated in FIG. 25, Segments A andB) may potentially be a reflection of the higher binding affinity forVEGFR-2 by the monomeric Peg-V2Bshort as compared to the VEGFR2 bindingsubunit included in the pegylated, his-tagged 385A08-Fn-V2B-Cysconstruct (see Example 6 above).

In the A673 Ewing Sarcoma xenograft model, pegylated, his-tagged385A08-Fn-V2B-Cys showed antitumor activity that was slightly weakerthan mono-specific ¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQ ID NO:28) (TGI=72.4% vs 82%, respectively), as shown in FIG. 26. This islikely a consequence of the lower affinity (approximately 4-5 foldlower) of the anti-VEGFR2 domain of pegylated, his-tagged385A08-Fn-V2B-Cys as compared to Peg-V2Bshort (see Example 6 above). Onthe other hand, the antitumor activity in the combo group was comparableto that of Peg-V2Bshort (TGI=80.2 vs 82%, respectively), suggesting thatthe A673 Ewing Sarcoma xenograft model is insensitive to IGF-IRinhibition because the IGF-IR inhibition contribution to the overallantitumor activity was rather marginal, as evidenced by the response tothe mono-specific ¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ ID NO: 27)administration (TGI=24.7%). Antitumor activities from pegylated,his-tagged 385A08-Fn-V2B-Cys at 100 mg/kg, as well as Peg-V2Bshort andthe combination of Peg-V2B short+AT580-PEG40 were significantlydifferent from vehicle. Additionally, there appeared to be a doseresponse between the two different doses tested for pegylated,his-tagged 385A08-Fn-V2B-Cys (50 vs 100 mg/kg) at the end of study (Day18, TGI=56.8% vs 72.4%, respectively), but without reachingstatistically significant differences between the two (FIG. 26).

Pegylated, his-tagged 385A08-Fn-V2B-Cys dose response was also evaluatedin the A673 Ewing Sarcoma model. The antitumor activity of doses between20 mg/kg and 200 mg/kg were compared; this dose range was selected tomaximize the likelihood to differentiate minimum vs. maximum efficaciousdoses. The different doses of pegylated, his-tagged 385A08-Fn-V2B-Cysdid not display a dose-dependent antitumor response. In fact, doses of20, 60, and 100 mg/kg were not distinguishable from each other(TGI=58.3, 67.1, 63.1, respectively) and 200 mg/kg had the maximuminhibitory activity (TGI=80.5%).

Example 10: PK/PD Studies on Multivalent Fibronectin Based Proteins

Single-Dose Pharmacokinetic Study with His-Tagged 385A08-Fn-V2B-Cys inMice.

FIG. 27 summarizes the pharmacokinetic parameters of his-tagged385A08-Fn-V2B-Cys in mice. Following a single IV dose of 5 or 50 mg/kg,his-tagged 385A08-Fn-V2B-Cys serum concentrations exhibited a slightlybi-exponential decline. The CLTp of his-tagged 385A08-Fn-V2B-Cys was0.11-0.12 mL/min/kg. The Vss (0.10-0.12 L/kg) was greater than plasmavolume but lower than the volume of extracellular fluid (0.2 L/kg). TheMRT and T1/2 of his-tagged 385A08-Fn-V2B-Cys were 15.2-16.8 and13.0-21.4 h, respectively.

After a single IP dose of 5 or 50 mg/kg, his-tagged 385A08-Fn-V2B-Cyswas rapidly absorbed (Tmax=1.4-3.0 h), with a serum concentration-timeprofile parallel to that after IV dosing. The absorption was nearlycomplete, with an absolute IP bioavailability of 83.1-105.8%. Theaverage Cmax at 5 and 50 mg/kg was 1.5 and 14.4 μM, respectively.

Single-Dose Pharmacokinetic/Pharmacodynamic Study with His-Tagged385A08-Fn-V2B-Cys in Nude Mice Bearing the Rh41 Tumor.

Following IP doses of 20 and 200 mg/kg of his-tagged 385A08-Fn-V2B-Cysto nude mice bearing the Rh41 tumor, the mVEGF-A levels showed dose- andtime-dependent increases in plasma (FIG. 28). Using an indirect-responsePD model, the plasma IC50 of his-tagged 385A08-Fn-V2B-Cys that inhibitedthe clearance of mVEGF-A from plasma by blocking its binding to VEGFR-2was estimated to be 13.8 μM at 200 mg/kg. At 20 mg/kg, the dynamic rangeof mVEGF-A concentrations may be too low to yield meaningful estimatesof PD parameters.

Single-Dose Pharmacokinetic/Pharmacodynamic Study with 385A08-Fn-V2B-Cys(Non-His Tagged) in Nude Mice Bearing the A673 Tumor.

Consistent with findings observed in nude mice bearing the Rh41 tumor,the mVEGF-A levels showed dose- and time-dependent increases in plasmafollowing IP doses of 20 and 200 mg/kg of 385A08-Fn-V2B-Cys (non-histagged) to nude mice bearing the A673 tumor FIG. 29. Using anindirect-response PD model, the plasma IC50 of 385A08-Fn-V2B-Cys(non-his tagged) that inhibited the clearance of mVEGF-A from plasma byblocking its binding to VEGFR-2 was estimated to be 7.0 μM at 200 mg/kg.At 20 mg/kg, the dynamic range of mVEGF-A concentrations may be too lowto yield meaningful estimates of PD parameters.

Single-Dose Pharmacokinetic/Pharmacodynamic Study with His-Tagged385A08-Fn-V2B-Cys in Monkeys.

FIG. 30 summarizes the pharmacokinetic parameters of his-tagged385A08-Fn-V2B-Cys in the monkey following a single IV dose of 3 or 30mg/kg. His-tagged 385A08-Fn-V2B-Cys plasma concentrations exhibited amono- or bi-exponential decline after the IV dose, with a significantdrop in concentrations at the terminal phase. The drop in plasmaconcentrations of his-tagged 385A08-Fn-V2B-Cys was presumably due to theformation of neutralizing antibodies that were detected in plasmasamples of the study. As a result, the plasma concentrations at the lasttime point were not included in the non-compartmental analysis.

In monkeys, his-tagged 385A08-Fn-V2B-Cys exhibited a slightdose-dependency in pharmacokinetics between 3 and 30 mg/kg. The CLTp at3 and 30 mg/kg was 0.029 and 0.023 mL/min/kg, respectively. The Vssranged from 0.056 to 0078 L/kg, greater than plasma volume. The MRT andT1/2 of his-tagged 385A08-Fn-V2B-Cys at 30 mg/kg were 57.9 and 42.7 h,respectively, slightly longer than those observed at 3 mg/kg (34.5 and23.2 h, respectively).

PK/PD modeling was conducted to understand the relationship between theplasma concentrations of his tagged 385A08-Fn-V2B-Cys and the elevationof circulating hVEGF-A levels. To fit the drop of plasma concentrationsof his tagged 385A08-Fn-V2B-Cys in the terminal phase, an addedclearance mechanism due to neutralizing antibodies was included in thePK model. The fitted vs. observed PK profiles are shown in FIG. 31. Theplasma IC50 of his tagged 385A08-Fn-V2B-Cys that inhibited the clearanceof hVEGF-A from plasma by blocking its binding to VEGFR-2 was estimatedto be 77-349 nM in monkeys, with a grand mean of 228 nM.

Single-Dose Pharmacokinetic/Pharmacodynamic Study with 385A08-Fn-V2B(Non-His Tagged) in Monkeys.

FIG. 32 summarizes the pharmacokinetic parameters of 385A08-Fn-V2B(non-his tagged) in the monkey following a 3 mg/kg IV dose as well asre-dosing the same monkeys at the same dose. Similar to his tagged385A08-Fn-V2B-Cys, there was a drop in the plasma concentrations of385A08-Fn-V2B (non-his tagged) at the terminal phase after the firstdose. As a result, the plasma concentrations at the last time point werenot included in the non-compartmental analysis. However, when385A08-Fn-V2B (non-his tagged) was re-dosed to the same monkeys, theeffect of antibodies on the pharmacokinetics of 385A08-Fn-V2B (non-histagged) appeared not to be significant (FIG. 32). In addition, thepharmacokinetics 385A08-Fn-V2B (non-his tagged) was comparable to thatof his tagged 385A08-Fn-V2B-Cys in monkeys.

Similar to what was done to his tagged 385A08-Fn-V2B-Cys, PK/PD modelingwas also conducted to understand the relationship between the plasmaconcentrations of 385A08-Fn-V2B (non-his tagged) and the elevation ofcirculating hVEGF-A levels. The fitted vs. observed PK profiles areshown in FIG. 33. The plasma IC50 of 385A08-Fn-V2B (non-his tagged) thatinhibited the clearance of hVEGF-A from plasma by blocking its bindingto VEGFR-2 was estimated to be 68-231 nM in monkeys, with a grand meanof 159 nM. These results are consistent with those observed with histagged 385A08-Fn-V2B-Cys.

Materials and Methods

High Throughput Protein Production (HTPP).

Selected binders containing a His₆ tag (SEQ ID NO: 59) were cloned intothe pET9d vector, transformed into E. coli HMS174 cells, inoculated into5 ml LB medium containing 50 μg/mL kanamycin in a 24-well format andgrown at 37° C. overnight. Fresh 5 ml LB medium (50 μg/mL kanamycin)cultures were prepared for inducible expression by aspirating 200 μlfrom the overnight culture and dispensing it into the appropriate well.The cultures were grown at 37° C. until A₆₀₀ 0.6-0.9. After inductionwith 1 mM isopropyl-β-thiogalactoside (IPTG), the culture was expressedfor 6 hours at 30° C. and harvested by centrifugation for 10 minutes at2,750×g at 4° C. Cell Pellets were frozen at −80° C.

Cell pellets (in a 24-well format) were lysed by resuspension in 4501 μlof Lysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ ProteaseInhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mMImidazole, 1 mg/ml lysozyme, 30 ug/ml DNAse, 2 ug/ml aprotonin, pH 8.0)and shaken at room temperature for 1-3 hours. Lysates were clarified andre-racked into a 96-well format by transfer into a 96-well Whatman GF/DUnifilter fitted with a 96-well, 1.2 mL catch plate and filtered bypositive pressure. The clarified lysates were transferred to a 96-wellNi-Chelating Plate that had been equilibrated with equilibration buffer(50 mM NaH₂PO₄, 0.5 M NaCl, 40 mM Imidazole, pH 8.0) and were incubatedfor 5 min. Unbound material was removed by vacuum. The resin was washed2×0.3 ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5 mMCHAPS, 40 mM Imidazole, pH 8.0) with each wash removed by vacuum. Next,the resin was washed with 3×0.3 ml/well with PBS with each wash stepremoved by vacuum. Prior to elution, each well was washed with 50 μlElution buffer (PBS+20 mM EDTA), incubated for 5 min, and the washdiscarded by vacuum. Protein was eluted by applying an additional 100 ulof Elution buffer to each well. After a 30 minute incubation at roomtemperature, the plate(s) were centrifuged for 5 minutes at 200 g.Eluted protein was collected in 96-well catch plates containing 5 μl of0.5 M MgCl₂ added to the bottom of the elution catch plate prior toelution. Eluted protein was quantified using a BCA assay with SGE as theprotein standard.

Midscale Expression and Purification of Insoluble Fibronectin-BasedScaffold Protein Binders.

For expression, selected clone(s), followed by the His6 tag (SEQ ID NO:59), were cloned into a pET9d (EMD Biosciences, San Diego, Calif.)vector and were expressed in E. coli HMS174(DE3) cells. Twenty ml of aninoculum culture (generated from a single plated colony) was used toinoculate 1 liter of LB medium containing 50 ag/mL kanamycin. Theculture was grown at 37° C. until A₆₀₀ 0.6-1.0. After induction with 1mM isopropyl-β-thiogalactoside (IPTG) the culture was grown for 6 hoursat 30° C. and was harvested by centrifugation for 30 minutes at ≥10,000g at 4° C. Cell Pellets were frozen at −80° C. The cell pellet wasresuspended in 25 mL of lysis buffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1×Complete™ Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, pH7.4) using an Ultra-turrax homogenizer (IKA works) on ice. Cell lysiswas achieved by high pressure homogenization (≥18,000 psi) using a ModelM-110S Microfluidizer (Microfluidics). The insoluble fraction wasseparated by centrifugation for 30 minutes at 23,300×g at 4° C. Theinsoluble pellet recovered from centrifugation of the lysate was washedwith 20 mM Sodium Phosphate/500 mM NaCl, pH 7.4. The pellet wasresolubilized in 6.0 M Guanidine Hydrochloride in 20 mM SodiumPhosphate/500 mM NaCl, pH 7.4 with sonication followed by incubation at37 degrees for 1-2 hours. The resolubilized pellet was filtered to 0.45um and loaded onto a HisTrap column equilibrated with the 20 mM SodiumPhosphate/500 mM NaCl/6.0 M Guanidine, pH 7.4 buffer. After loading, thecolumn was washed to baseline UV A280 absorbance with 20 column volumes(CV) of equilibration buffer, followed by washing for an additional 25CV with the same buffer. Bound protein was eluted with 500 mM Imidazolein 20 mM Sodium Phosphate/500 mM NaCl/6.0 M Guan-HCl, pH 7.4. Thepurified protein was refolded by dialysis against 50 mM SodiumAcetate/150 mM NaCl pH 4.5.

Midscale Expression and Purification of Soluble Fibronectin-BasedScaffold Protein Binders.

As an alternative to purification of insoluble binders, the purificationof soluble binders may also be used. For expression, selected clone(s),followed by the His₆ tag (SEQ ID NO: 59), are cloned into a pET9d (EMDBiosciences, San Diego, Calif.) vector and are expressed in E. coliHMS174(DE3) cells. Twenty ml of an inoculum culture (generated from asingle plated colony) is used to inoculate 1 liter of LB mediumcontaining 50 μg/mL kanamycin. The culture is grown at 37° C. until A₆₀₀0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG)the culture is grown for 6 hours at 30° C. and is harvested bycentrifugation for 30 minutes at ≥10,000×g at 4° C. Cell Pellets arefrozen at −80° C. The cell pellet is resuspended in 25 mL of lysisbuffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) using an Ultra-turraxhomogenizer (IKA works) on ice. Cell lysis is achieved by high pressurehomogenization (≥18,000 psi) using a Model M-110S Microfluidizer(Microfluidics).

The soluble fraction is separated by centrifugation for 30 minutes at23,300 g at 4° C. The supernatant is clarified via 0.45 μm filter. Theclarified lysate is loaded onto a HisTrap column (GE) pre-equilibratedwith 20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4. The column is then washed with25 column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4, followed by 20column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 25 mM imidazole pH 7.4, andthen 35 column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole pH7.4. Protein is eluted with 15 column volumes of column volumes of 20 mMNaH₂PO₄, 0.5 M NaCl, 500 mM imidazole pH 7.4, fractions are pooled basedon absorbance at A₂₈₀ and are dialyzed against 1×PBS, 50 mM Tris, 150 mMNaCl, pH 8.5 or 50 mM NaOAc; 150 mM NaCl; pH4.5. Any precipitate isremoved by filtering at 0.22 μm.

Large scale expression and purification of the fibronectin-basedscaffold protein binders was also used. Other than the quantitiesproduced, this method is substantially similar to the insoluble midscalepurification methods.

Biophysical Characterization of Fibronectin-Based Scaffold ProteinBinders

Size Exclusion Chromatography (SEC).

Standard size exclusion chromatography (SEC) was performed on theproteins purified from the HTPP, midscale processes, and large scaleprocesses (0.1 to 1 μg of protein for HTPP and 10-50 μg for midscale).SEC of HTPP derived material was performed using a Superdex 200 5/150column (GE Healthcare) or on a Superdex 200 10/30 column (GE Healthcare)for midscale material on an Agilent 1100 or 1200 HPLC system with UVdetection at A₂₁₄ nm and A₂₈₀ nm and with fluorescence detection(excitation=280 nm, emission=350 nm). A buffer of 100 mM sodium sulfate,100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8 at appropriateflow rate of the SEC column was employed. Gel filtration standards(Bio-Rad Laboratories, Hercules, Calif.) were used for molecular weightcalibration.

Mass Spectrometry.

The midscale and large scale purified ¹⁰Fn3-based binders were furtheranalyzed by LC-MS (Water's 2695 liquid chromatography HPLC systemcoupled with Waters Q-TOF API mass spectrometer, Waters Corporation,Milford, Mass.). Samples were diluted to approximately 0.5 mg/ml withHPLC grade water. Approximately 5 μl of diluted sample was injected ontoa Jupiter C18 column (Catalog number 00G-4053-80, Phenomenex). Buffer A:0.02% TFA+0.08% formic acid in HPLC grade water. Buffer B: 0.02%TFA+0.08% formic acid in HPLC grade acetonitrile. Sample was eluted withgradient (Table 1) at a flow rate of 0.2 ml/minutes.

Differential Scanning Calorimetry (DSC).

Differential Scanning Calorimetry (DSC) analysis of the midscale andlarge scale purified ¹⁰Fn3-based binders was performed to determine theT_(m). A 1 mg/ml solution was scanned in a N-DSC II calorimeter(Calorimetry Sciences Corp) by ramping the temperature from 5° C. to 95°C. at a rate of 1 degree per minute under 3 atm pressure. The data wasanalyzed versus a control run of the appropriate buffer using a best fitusing Origin Software (OriginLab Corp).

Determination of Binding Affinity Using Surface Plasmon Resonance(BIAcore) Analysis

Selected HTPP purified fibronectin based scaffold proteins, and selectedmidscale and large scale versions of the tandem construct 385A08-Fn-V2B(pegylated and non-pegylated versions; his-tag and non-his-tag versions)were evaluated for their kinetic behavior towards IGF-IR using surfaceplasmon resonance. A capture assay was developed utilizing a humanIGF-IR-Fc fusion. A similar reagent had been described by Forbes et al.(Forbes et al. 2002, European J. Biochemistry, 269, 961-968). Theextracellular domain of human IGF-IR (aa 1-932) was cloned into amammalian expression vector containing the hinge and constant regions ofhuman IgG. Transient transfection of the plasmid produced a fusionprotein, IGF-IR-Fc as described next. 293T cells (a human embryonickidney cell line expressing SV40 large T antigen) were obtained fromGenehunter (Nashville, Tenn.) and maintained according to themanufacturer's instructions. Briefly, 12×10⁶ cells were seeded in T175flasks (Falcon) for transfection with the appropriate DNA preparations(Qiagen, Valencia, Calif.) and Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) and Optimem (Invitrogen, Carlsbad, Calif.). Conditioned mediawas collected at 72 hrs and expression verified by Western blottingusing anti-human IgG-HRP (Pierce, Rockford, Ill.) and anti-IGF-IRpolyclonal antibody (R&D Systems, Minneapolis, Minn.). This IGF-IR-Fcfusion protein was subsequently purified by Protein A chromatography.

One method of obtaining kinetic measurements, which was used for theHTPP purified constructs (non-pegylated, his tag versions) was tocapture the IGF-IR-Fc on Protein A/G immobilized on Biacore CM5 chips(GE, Piscataway, N.J.) by amine coupling. The kinetic analysis involvedthe capture of IGF-IR-Fc on Protein A/G followed by injection of aconcentration series of the fibronectin based scaffold proteins insolution and regeneration of the Protein A/G surface by glycine pH 2.0.Protein concentrations from HTPP material are approximate, thereforeK_(D) determinations are also approximate. Sensorgrams were obtained ateach concentration and fitted via Biaevaluation to determine the rateconstants k_(a) (k_(on)) and k_(d) (k_(off)). The dissociation constant,K_(D), was calculated from the ratio of rate constants k_(off)/k_(on).

An alternative method is to capture the IGF-IR-Fc on Protein A. Thismethod was used for calculating the affinity and kinetics of certainversions of the construct 385A08-Fn-V2B-cys (both pegylated andnon-pegylated and with or without his tag) from midscale and large scalematerials. Specifically, IGF-IR-Fc was captured on Protein A immobilizedon a Biacore CM5 sensor chip by amine coupling. VEGFR2-Fc wasimmobilized directly on a CM5 chip by amine coupling. The affinity andkinetic analysis involved the capture of IGF-IR-Fc on Protein A followedby injection of the tandem adnectin in solution over IGF-IR-Fc andVEGFR2-Fc. The analytical surface was regenerated between samples withglycine pH 1.5. Sensorgrams were obtained at each concentration and wereevaluated using Biacore T100 evaluation software to determine the rateconstants k_(a) (k_(on)) and k_(d) (k_(off)).

Another method to assess the functionality of the IGF-IR domain is tocapture the IGF-IR-Fc on anti-human IgG (GE, Piscataway, N.J.). This wasdone for the midscale purified construct 385A08-Fn-V2B (non-pegylated,his-tag) as described in Example 5 above. The anti-human IgG wasimmobilized on flow cells 1 and 2 of a CM5 chip surface according themanufacturer's instructions. 100 nM human IGF-IR-Fc was injected for 2minutes at 10 ul/min on flow cell 2. Subsequently a concentration seriesof the midscale prep was injected across both flow cell surfaces at 50uL/min. Two injections of 50 mM Glycine pH 1.7 were used to regeneratethe surface.

The functionality of the V2B domain was assessed by evaluating aconcentration series of the midscale and large scale purification of thetandem construct 385A08-Fn-V2B (pegylated, and non-pegylated versions,his-tag and non-his-tag versions) vs. VEGFR2-Fc that was directlyimmobilized on a CM5 chip surface (GE, Piscataway, N.J.). VEGFR2-Fc (R&DSystems, MN) was diluted to 12 ug/mL in 50 mM Acetate pH 5.0 for animmobilization level of ˜1300 RU. Varying concentrations of385A08-Fn-V2B in the solution phase were monitored for association (2minutes) and dissociation (10 minutes) kinetics at a flow rate of 50uL/min. Two 30 second injections of 50 mM glycine pH 1.7 were used toregenerate the surface.

Pegylation

Multi-valent fibronectin based scaffold proteins, such as V/I¹⁰Fn3-based binders, can be pegylated with various sizes and types ofPEG. To allow for pegylation, the protein is typically modified near theC-terminus by a single point mutation of an amino acid, typically aserine, to a cysteine. PEGylation of the protein at the single cysteineresidue is accomplished by conjugating various maleimide-derivatized PEGforms, combining the PEG reagent with the protein solution andincubating. Confirmation of the PEGylation of the protein can beconfirmed by SDS-Page and/or SE-HPLC methods that can separate thenon-PEGylated protein from the PEGylated protein.

For example, the construct 385A08-Fn-V2B was pegylated by replacing aserine that was at position 203 with a cysteine. The resultingconstruct, 385A08-Fn-V2B-Cys, was then conjugated with a 40 kD PEG. ThePEG reagent was mixed with the protein construct (385A08-Fn-V2B-Cys) insolution and incubated. Studies to confirm the pegylation were alsoconducted as described in the paragraph above. Studies were alsoconducted on the tandem construct with linear PEG. In addition, thistype of pegylation can also be done with a his-tagged protein.

Additionally, the linear and branched pegylated versions of theconstruct 385A08-Fn-V2B-Cys were used to conduct certain pharmacokineticstudies in mice to assess the difference between linear vs. branchedPEG. Both were 40 kD PEGs, and the linear version of this construct alsoincluded a his-tag.

V/I ¹⁰Fn3-based binders may also be pegylated using an alternativemethod. Five ml of an inoculum culture of BL21(DE3) E. coli cellscontaining a T7 ploymerase driven pET29 plasmid encoding a V/I¹⁰Fn3-based binder is generated from a single plated colony and used toinoculate 1 liter of auto-induction media (“ONE” medium, EMDBiosciences, San Diego, Calif.) containing 50 μg/mL kanamycin.Expression is carried out at 18° C. after initial growth at 37° C. andharvested by centrifugation for 10 minutes at ˜10,000×g at 4° C. Cellpellets are frozen at 80° C. The cell pellet is resuspended in 10 mL oflysis buffer (20 mM NaH₂PO₄, 0.5 M NaCl, 5 mM Immidazole, pH 7.4) andmechanically lysed using an Avestin homgenizer. The soluble fraction isseparated by centrifugation for 15 minutes at 23,300×g at 4° C. Thesupernatant is decanted and the pellet is solubilized in Lysis buffer(above) supplemented with 4 M to 6 M guanidine hydrochloride (GdnHCl).Solubilized protein is then purified on a suitably sized NiNTA column(Qiagen, Inc.) pre-equilibrated with the GdnHCL supplemented LysisBuffer. The column is then washed with 5 to 10 column volumes of thesame buffer, followed by elution with the same buffer supplemented with300 mM Immidazole. The fractions eluted off the column containing theprotein of interest are diluted to 2-3 mgs/mL protein and then combinedwith a 1.2-1.5 molar excess of solid NEM-PEG (40 kDa branched or other).The mixture is allowed to react at room temperature for 30 minutes oruntil the reaction is complete. The entire reaction volume is thenplaced into a dialysis bag (5,000 Da Molecular Weight cutoff) and themixture is subjected to a dialysis refolding process. The dialysate fromthis procedure contains properly folded, PEGylated materials plus excessreactants. The mixture of products and excess reactants from thePEGylation reaction are clarified via centrifugation or filtration priorto loading them onto a cation exchange chromotography column (SPSepharose or Resource S, GE Healthcare). The column is developed with150 mM to 1 M NaCl gradient in the NaOAc background buffer. Studies toconfirm the pegylation are conducted as described above.

Competitive Blocking Assays

R⁺ Competitive Blocking Assay.

R⁺, a gift from Renato Baserga (Thomas Jefferson University,Philadelphia, Pa.), is a mouse embryo fibroblast overexpressing humanIGF-IR in the context of a deletion in the mouse IGF-IR gene. Cells wereplated in 96 well plates at a concentration of 20,000 cells per well inDMEM (Invitrogen, Carlsbad, Calif.) containing 5% calf serum (Hyclone,Logan, Utah). The following day, cells were washed twice in serum freeDMEM containing 0.1% BSA (Invitrogen, Carlsbad, Calif.). After thewashes, the cells were incubated at 4° C. for 10 minutes in bindingbuffer (serum free DMEM+0.1% BSA) containing increasing concentrationsof IGF-IR antagonists or controls. Typical controls included themono-specific ¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ ID NO: 27)(positive control) or the mono-specific ¹⁰Fn3-based VEGFR2 binder(Peg-V2Bshort, SEQ ID NO: 28) (negative control). Following this briefexposure to antagonists, R⁺ cells were additionally exposed to 200 nM ofa bi-valent ¹⁰Fn3-based IGF-IR binder (AT580-PEG20-AT580, SEQ ID NO: 27)labeled with IRDye 800CW according to the manufacturer instructions(Li-Cor Biosciences, Lincoln, Nebr.). After incubation for 4 hours at 4°C., cells were washed twice in binding buffer and visualized on theOdyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, Nebr.).The resulting data was analyzed using GraphPad Prism (GraphPad Software,La Jolla, Calif.).

293:KDR Competitive Blocking Assay.

293:KDR (Sibtech, Brookfield, Conn.) were plated in 96 well plates at aconcentration of 25,000 cells per well in DMEM (Invitrogen, Carlsbad,Calif.) containing 10% fetal bovine serum (Hyclone, Logan, Utah). Thefollowing day, cells were washed twice in serum free DMEM containing0.1% BSA (Invitrogen, Carlsbad, Calif.). After the washes, the cellswere incubated at 4° C. for 10 minutes in binding buffer (serum freeDMEM+0.1% BSA) containing increasing concentrations of VEGFR-2antagonists or controls. Typical controls included the mono-specific¹⁰Fn3-based VEGFR2 binder (Peg-V2Bshort, SEQ ID NO: 28) (positivecontrol) or the mono-specific ¹⁰Fn3-based IGF-IR binder (AT580-PEG40,SEQ ID NO: 27) (negative control). Following this brief exposure toantagonists, 293:KDR cells were additionally exposed to 200 nMPeg-V2Bshort labeled with IRDye 800CW according to the manufacturerinstructions (Li-Cor Biosciences, Lincoln, Nebr.). After incubation for4 hours at 4° C., cells were washed twice in binding buffer andvisualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences,Lincoln, Nebr.). The resulting data was analyzed using GraphPad Prism(GraphPad Software, La Jolla, Calif.).

In Vitro Proliferation Assays

RH41. RH41 cells were grown in RPMI medium supplemented with 10% fetalbovine serum, 10 mM Hepes, glutamax, penicillin and streptomycin. Cellproliferation was evaluated by [³H]-thymidine incorporation orcolorimetric methods. For evaluation of [³H]-thymidine incorporation,cells were plated at an optimized density (4K cells/well) in 96-wellplates, incubated overnight at 37° C., then exposed to a serial dilutionof the fibronectin based scaffold proteins. After 72 hours incubation,cells were pulsed with 4 μCi/ml [³H]-thymidine (Amersham PharmaciaBiotech, UK) for 3 hours, trypsinized, harvested onto UniFilter-96 GF/Bplates (PerkinElmer, Boston, Mass.) and scintillation was measured on aTopCount NXT (Packard, Conn.). Results were expressed as an IC50, whichis the drug concentration required to inhibit cell proliferation by 50%compared to untreated control cells. The mean IC50 and standarddeviation from multiple tests for each cell line were calculated. Forcolorimetric evaluation, cells were plated in 96 well plates at aconcentration of 5,000 cells per well in 90 ul/well of RPMI-glutamax(Invitrogen, Carlsbad, Calif.) containing 10% fetal bovine serum(Hyclone, Logan, Utah) and incubated for 24 hours, 37° C., 5% CO₂. 10 ulof 10× concentrations of ¹⁰Fn3-based IGF-IR antagonists were added tothe wells and incubated for 72 hours at 37° C., 5% CO₂. After theproliferation period, cells were exposed to Cell Titer 96 AqueousProliferation Reagent (Promega, Madison, Wis.) and allowed to incubatefor an additional four hours. Absorbance at 490 nm was measured on aSpectramax Plus 384 (Molecular Devices, Sunnyvale, Calif.), and theresulting data was analyzed using Softmax Pro 5 software (MolecularDevices, Sunnyvale, Calif.).

Ba/F3.

Murine Ba/F3 cells stably expressing a VEGFR2 fusion protein (comprisingthe extracellular domain of hVEGFR2 and the intracellular domain ofhEpoR) were plated in 96-well plates at 25,000 cells/well in 90 uLgrowth media containing 15 ng/mL VEGF-A. Serial dilutions of fibronectinscaffold domain proteins were prepared at 10× final concentration, and10 uL of the protein was added to each well. Plates were incubated at37° C./5% CO₂ for 48-72 hours. Cell proliferation assay reagent(CellTiter 96, Promega) was added to each well (20 μL/well), and theplates were further incubated for 3-4 hours. At the end of theincubation period, absorbance was read (A490) in a 96-well plate reader.

HMVEC-L.

Primary human microvascular endothelial cells from Lung (HMVEC-L), werepurchased from Lonza (Cat# CC-2527; Walkersville, Md.) and maintained inEGM-2 MV Singlequots (Lonza, Cat# CC-3202), then switched toRPMI-1640+Glutamax supplemented with 2% Heat Inactivated fetal calfserum (FCS), 10 mM Hepes, 15 ng/ml recombinant Human VEGF (Biosource),and 50 ng/ml recombinant Human IGF-1 (PeproTech) for the growth assay.Proliferation was evaluated by incorporation of [³H]-thymidine into DNAafter exposure of cells to compounds in the presence of 10% Fetal CalfSerum (FCS). Typical controls included the following: IGF-IR monoclonalantibody mAB391 (R & D Systems, Minneapolis, Minn.), a mono-specific¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ ID NO: 27), a mono-specific¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQ ID NO: 28), and a wildtype ¹⁰Fn3-based protein that does not bind to a target (SGE). HMVEC-Lcells were plated at 1,500 cells/well in 96-well microtiter Falconplates (cell density was optimized for this cell type). After 72 hoursincubation at 37° C., cells were pulsed with 4 μCi/ml [6⁻³H]thymidine(Amersham Pharmacia Biotech, UK) for 3 hours, trypsinized, harvestedonto UniFilter-96 GF/B plates (PerkinElmer, Boston, Mass.), andscintillation counts were measured on a TopCount NXT (Packard, Conn.).Results are expressed as the drug concentration required for inhibitionof cellular proliferation by 50% to that of untreated control cells(IC₅₀).

NCI-H929.

NCI-H929 (ATCC, Manassas, Va.), an IGF-dependent human plasmacytoma cellline, was plated in 96 well plates at a concentration of 25,000 cellsper well in DMEM (Invitrogen, Carlsbad, Calif.) containing 10% fetalbovine serum (Hyclone, Logan, Utah) in the presence of IGF-IRantagonists or controls. Typical controls included the following: IGF-IRmonoclonal antibody MAB391 (R&D Systems, Minneapolis, Minn.), amono-specific ¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ ID NO: 27), amono-specific ¹⁰Fn3-based VEGFR-2 binder (Peg-V2Bshort, SEQ ID NO: 28),and a wild type ¹⁰Fn3-based protein that does not bind to a target(SGE). Cells were allowed to proliferate for 72 hours at 37° C., 5% CO₂.After the proliferation period, cells were exposed to Cell Titer 96Aqueous Proliferation Reagent (Promega, Madison, Wis.) and allowed toincubate for an additional four hours. Absorbance at 490 nm was measuredon a Spectramax Plus 384 (Molecular Devices, Sunnyvale, Calif.), and theresulting data was analyzed using GraphPad Prism (GraphPad Software, LaJolla, Calif.).

Cell-Based Signal Transduction Assays

HEK293/KDR and PAE/KDR Assays.

HEK293/KDR and PAE/KDR cells (Sibtech, Inc) were cultured in DMEM withGlutaMAX™-1 (Gibco, Carlsbad, Calif.) containing 10% fetal bovine serum(FBS), penicillin, streptomycin, HEPES and puromycin, and grown to ˜70%confluency. Cells were placed into starvation medium (DMEM-GlutaMAX,0.5% FBS, penicillin, streptomycin, HEPES and puromycin) overnight at37° C., then stimulated with VEGF or IGF-1 (50 ng/ml, PeproTech, RockyHill, N.J.) for 10 min at 37° C. Unstimulated cells were included ascontrols. Cells were rinsed twice with ice-cold PBS on ice and extractswere prepared in TTG lysis buffer (1% Triton X-100, 5% glycerol, 0.15 MNaCl, 20 mM Tris-HCl pH 7.6, Complete tablet (Roche, Indianapolis, Ind.)and Phosphatase Inhibitor Cocktail 2 (Sigma, Milwaukee, Wis.). Proteinconcentrations of total cell lysates were determined using a BCA assaykit (Pierce, Rockford, Ill.). Lysates (30 μg) were resolved by SDS-PAGE(Invitrogen, Carlsbad, Calif.), transferred to nitrocellulose membranes(Bio-Rad Laboratories, Hercules, Calif.) and immunoblotted withantibodies to phospho VEGFR (Tyr 996) (Santa Cruz Biotechnology,Carlsbad, Calif.), phospho IGF1R/IR (Tyr1135/1136)/Insulin Receptor(Tyr1150/1151), phospho Akt (Ser 473), phospho p44/42MAPK(Thr202/Tyr204) (Cell Signaling Technology, Beverly, Mass.) or totalActin (Chemicon International, Temecula, Calif.) in Odyssey BlockingBuffer with 0.1% Tween 20 (Li-Cor Biosciences, Lincoln, NB). Membraneswere incubated with the appropriate infrared-labeled secondaryantibodies from Rockland Immunochemicals, Inc. (Gilbertsville, Pa.) andMolecular Probes (Carlsbad, Calif.). Protein visualization was performedusing Li-Cor Biosciences Odyssey Infrared Imaging System.

HEK293/KDR cells (Sibtech, Inc) were cultured as described above.VEGFR-IGF-IR multivalent fibronectin scaffold domains were diluted instarvation media and added to cells at a final concentration of 100 and10 nM for 1 hour at 37° C. Cells were stimulated with VEGF-IGF-1 ligandcombination (both 50 ng/ml final concentration, PeproTech, Rocky Hill,N.J.) for 10 min at 37° C. Unstimulated cells were included as controls.Cell lysates were prepared as described above. Lysates (30 μg) wereresolved by SDS-PAGE (Invitrogen, Carlsbad, Calif.), transferred tonitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.) andprobed with antibodies to total VEGFR, phospho VEGFR (Tyr 996), totalIGF-1R (Santa Cruz Biotechnology, Carlsbad, Calif.), phosphoIGF1R/IR(Tyr11135/1136)/Insulin Receptor (Tyr1150/1151), total Akt,phospho Akt (Ser 473), total MAPK, phospho 44/42 MAPK(Thr202/Tyr204)(Cell Signaling Technology, Beverly, Mass.) or Actin (ChemiconInternational, Temecula, Calif.). Antibodies were detected afterincubation with infrared-labeled secondary antibodies using Li-CorBiosciences Odyssey Infrared Imaging System.

PAE/KDR cells (Sibtech, Inc) were cultured as described above.VEGFR-IGF-IR multivalent fibronectin scaffold domains were diluted instarvation media and added to cells at a final concentration of 100 and10 nM for 1 hour at 37° C. Cells were stimulated with VEGF-IGF-1 ligandcombination (both 50 ng/ml final concentration, PeproTech, Rocky Hill,N.J.) for 10 min at 37° C. Unstimulated cells were included as controls.Cell lysates were prepared as described above. Lysates (30 μg) wereresolved by SDS-PAGE (Invitrogen, Carlsbad, Calif.), transferred tonitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.) andprobed with antibodies to total VEGFR, phospho VEGFR (Tyr 996), totalIGF-1R (Santa Cruz Biotechnology, Carlsbad, Calif.), phosphoIGF1R/IR(Tyr11135/1136)/Insulin Receptor (Tyr1150/1151), total Akt,phospho Akt (Ser 473), total MAPK, phospho 44/42 MAPK(Thr202/Tyr204)(Cell Signaling Technology, Beverly, Mass.) or Actin (ChemiconInternational, Temecula, Calif.). Antibodies were detected afterincubation with infrared-labeled secondary antibodies using Li-CorBiosciences Odyssey Infrared Imaging System.

Western Blot Analysis to Measure Signaling Pathways Inhibited in HMVEC-LCells or Rh41 Cells

HMVEC-L cells were cultured in EGM-2MV Singlequots complete media(Lonza, Cat# CC-3202), and grown to ˜70% confluence. Cells were placedinto starvation medium (Basal EBM-2 media, 0.3% BSA) for 6 hrs, 37° C.,5% CO₂. Test reagents were diluted in starvation media and added tocells at a final concentration of 100 nM for 1 hr at 37° C. Cells werestimulated with a combination of VEGF and IGF-1 ligands (50 ng/ml each),(VEGF, R&D Systems, #293-VE/CF; IGF1, PeproTech, Rocky Hill, N.J.,#100-11) for 10 min at 37° C. Unstimulated cells served as controls.

Rh41 cells were cultured in RPMI (Invitrogen, Carlsbad, Calif.)containing 10% fetal bovine serum (FCS), penicillin, streptomycin, andHEPES, and grown to ˜70% confluence. Rh41 is a human rhabdomyosarcomatumor cell line which was obtained from Dr. Lee Helman (NIH), andmaintained in RPMI-1640+Glutamax, in the presence of 10% FCS, 10 mMHEPES; Rh41 cells are IGF-dependent and are very sensitive to inhibitionby antagonists of IGF-1R (i.e., small molecules, mononectins, andmAB391). Rh41 cells were placed into starvation medium (RPMI, 0.3% BSA)overnight at 37° C. Test reagents were diluted in starvation media andadded to cells at a final concentration of 100 nM for 1 hour at 37° C.Cells were stimulated with a combination of VEGF and IGF-1 ligands (50ng/ml each), for 10 min at 37° C. Typical controls included thefollowing: Unstimulated cells, IGF-1R monoclonal antibody mAB391 (R & DSystems, Minneapolis, Minn.), a mono-specific ¹⁰Fn3-based IGF-IR binder(AT580-PEG40, SEQ ID NO: 27), or a mono-specific ¹⁰Fn3-based VEGFR-2binder (Peg-V2Bshort, SEQ ID NO: 28).

HMVEC-L or Rh41 cells were rinsed twice with ice-cold PBS on ice andextracts were prepared in TTG lysis buffer (1% Triton X-100, 5%glycerol, 0.15 M NaCl, 20 mM Tris-HCl pH 7.6, Complete Tablet (Roche,Indianapolis, Ind.) and Phosphatase Inhibitor Cocktail 2 (Sigma,Milwaukee, Wis.). Protein concentrations of total cell lysates weredetermined using a BCA assay kit (Pierce, Rockford, Ill.). Equal amountsof protein from each lysate (40 ug) were added to each well of a gel(NuPAGE 4-12% Bis-Tris Gel, Invitrogen, Carlsbad, Calif.). Proteins wereseparated on a NuPAGE gel, transferred to nitrocellulose membranes(Bio-Rad Laboratories, Hercules, Calif.) and incubated in OdysseyBlocking buffer (Li-Cor Biosciences) for 1 hr at room temperature.Inhibition of phosphorylation was measured by probing Western blots withantibodies specific for IGF-1R (Phospho-IGF-1Rβ, (Tyr1135/1136)/IR,(Tyr1150/1151) (19H7) Antibody, (Cell Signaling #3024)), IGF-1Rβ (SantaCruz Biotechnology, Inc. sc-713), Akt (pAkt (Ser473) (Cell Signaling#4051)), Akt (Cell Signaling #9272), MAPK (p-p44/42) MAPK(Thr202/Tyr2040) (E10) (Cell Signaling #9106), (MAPK Cell Signaling#9102), and VEGFR-2 (pFlk-1) (Tyr996)-R, (Santa Cruz Biotechnology, Inc.sc-16629R), VEGFR-2 (Flk-1) (C-1158), (Santa Cruz Biotechnology, Inc.sc-504), total GAPDH (Cell Signaling #2118) in Odyssey Blocking Bufferwith 0.1% Tween 20 (Li-Cor Biosciences, Lincoln, NB), for 3 hrs at roomtemperature. Membranes were washed 3 times in TBS with 0.1% Tween-20 andthen incubated with IR-labeled secondary antibodies for 1 hr at roomtemperature. Protein analysis was performed utilizing the OdysseyInfrared Imaging System (Li-Cor Biosciences) which enables simultaneousand independent detection of fluorescent signals.

Ca2+ Flux Assays

HMVEC-L cells were plated at 2.5×10⁴ cells/well on poly-D-Lysine-coated96-well plates (Falcon #356640) in EGM2-MV Singlequots complete media(Lonza, Cat#CC-3202). Cells were starved overnight in basal EBM media(Lonza). The day of the assay, test reagents were serial diluted in HHPbuffer (10 mM HEPES, 2.5 mM probenecid [Sigma #P8761] in HBSS [GibcoBRL#14025-076]+0.1% BSA) and added to the cells after aspirating EBM media.Calcium-4 dye kit (Molecular Devices, #R8142) was added at the same timeas the HHP buffer, and cells were incubated for 1 hour at 37° C. Ca2⁺release was triggered by the addition of 50 ng/ml VEGF (Invitrogen#PHG0145). Ca2⁺ flux was monitored by measurement on a FLIPR (MolecularDevices).

Human Microvascular Endothelial Cell Tube Formation

Matrigel™ plates were prepared by thawing Matrigel™ (BD Biosciences,#356237) overnight at 4° C. Matrigel™ (300 μl/well) was added to a24-well plate and subsequently incubated at 37° C. for 30 minutes inorder for the gel to polymerize. HMVEC-L cells were cultured in EGM-2MVcomplete media (Lonza, Cat# CC-3202), and grown to ˜70% confluence. Thecells were resuspended (1×10⁵ cells/ml) with test compounds (100 nM) orwithout compounds in EGM-2MV complete media. 1 ml of the cell suspensionwas added to each well. Plates were incubated for 12 hrs at 37° C., in5% CO₂. The growth medium was aspirated and 1 ml of 0.3% Glutaraldehyde(VWR, #GX015305-1) was added to each well at room temperature for 30minutes. The media was aspirated and cells were washed with 1 ml washbuffer (Cell Spreading Reagent Kit, Pierce, #K0600011). 500 μl ofpermeabilization solutions were added followed by incubation at roomtemperature for 15 minutes. The media was aspirated and cells werewashed with 1 ml wash buffer. 500 μl of stain solution was added to eachwell and incubated at room temperature in the dark for 1 hr. Thesolution was removed by aspiration and washed 3× with wash buffer. Theplates were sealed, and imaged on an ArrayScan HCS Reader. Theangiogenic index, which is a measure of the imaged area occupied bymicrovascular tubes, was automatically calculated and reported for eachwell.

Single-Dose PK in Mice

The pharmacokinetics of 385A08-Fn-V2B-Cys (SEQ ID NO: 9) pegylated with40 kD linear or 40 kD branched PEG was studied in mice. Two groups ofnon-fasted animals (N=3-4 per group, 20-25 g) received Adnectin as anintravenous (IV) bolus doses via the tail vein (5 mL/kg). The dose was50 mg/kg. Prior to dosing, the compound was diluted from a stocksolution to an appropriate dosing solution concentration in phosphatebuffered saline (PBS). Serial blood samples (˜0.05 mL) were obtained bynicking the lateral tail vein. For the IV route, the sampling timepoints were 0.08, 0.5, 1, 2, 6, 12, 24, 48, 96, and 171 h post dose.Plasma samples were harvested by diluting blood sample into a citratephosphate dextrose solution in a 1:1 ratio and were stored at −20° C.until analysis.

Single-Dose PK/PD Study in Rh41-Tumored Mice

Pharmacodynamic studies were conducted in Rh41 tumored-mice usingincreasing concentrations after a single dose of a pegylated, his-tagversion of construct 385A08-FnOV2B-Cys (50, 100, 200 mpk), or acombination of a mono-specific ¹⁰Fn3-based IGF-IR binder (AT580-PEG40,SEQ ID NO: 27) and a mono-specific ¹⁰Fn3-based VEGFR-2 binder(Peg-V2Bshort, SEQ ID NO: 28) at equivalent doses (25+25, 50+50,100+100). Tumors were harvested at 1, 6, and 24 hrs after dosing andprocessed. Equal amounts of protein from each tumor lysate (60 Gtg) wereadded to each well of a gel (NuPAGE 4-12% Bis-Tris Gel, Invitrogen,Carlsbad, Calif.). Proteins were separated on a NuPAGE gel, transferredto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.) andincubated in Odyssey Blocking buffer (Li-Cor Biosciences) for 1 hr atroom temperature. Inhibition of phosphorylation of proteins was measuredby probing Western blots with phospho-specific antibodies as describedabove.

Tumor Xenograft Studies RH-41, A549 and GEO, in Mice

Female Balb/c athymic mice (nu/nu), 5-6 weeks of age were purchased fromHarlan Sprague-Dawley Co. (Indianapolis, Ind.). Animals were maintainedin an ammonia and pathogen-free environment and fed water and food adlibitum. Mice were maintained in quarantine for 7 days prior to tumorimplantation and efficacy testing. All animal studies were performedunder the approval of the BMS Animal Care and Use committee and inaccordance with the American Association for Accreditation of LaboratoryAnimal Care (AAALAC).

Human tumors Rh-41 (rhabdomyosarcoma), A549 (lung) and GEO (colon) wereimplanted subcutaneously (sc) as small fragments generally no largerthan 0.1 to 0.2 mm³ using a 16 g trocar. All tumors that were evaluatedwere allowed to grow to an approximate size of 125 mm³ prior at theinitiation of treatment. The treatment and control group sizes consistedof 6-8 mice. Tumor size was measured twice weekly. Tumor volume wascalculated by measuring perpendicular tumor diameters using Vernierscale calipers and using the formula for an ellipsoid: ½(length×(width)²). Mice were euthanized if the tumor size surpassed1,500 mm³, if animal weight loss was greater than 20% of the startingweight or if extensive tumor necrosis occurred. All compounds wereobtained internally and dosed by intraperitoneal (i.p.) delivery using a25 g sterile hypodermic needle. Vehicle control solution was generallyphosphate buffered saline (PBS) while a mono-specific ¹⁰Fn3-basedVEGFR-2 binder (Peg-V2Bshort, SEQ ID NO: 28) was dosed in a formulationof 10 mM Sodium Acetate, 150 mM NaCl, pH 4.5; a mono-specific¹⁰Fn3-based IGF-IR binder (AT580-PEG40, SEQ ID NO: 27) was formulated inPBS and pegylated, his-tagged construct 385A08-Fn-V2B-cys (SEQ ID NO: 9)was formulated in 50 mM Sodium Acetate, 150 mM NaCl, pH, 4.5.

Anti-tumor efficacy is expressed as percent tumor growth inhibition (%TGI) and calculated as follows:% TGI={1−[(T _(t) −T _(o))/(C _(t) −C _(o))]}×100where C_(t)=the median tumor volume (mm³) of vehicle control (C) mice attime, t; T_(t)=median tumor volume (mm³) of treated mice (T) at time t;C₀=median tumor volume (mm³) of vehicle control (C) mice at time 0;T_(t)=median tumor volume (mm³) of treated mice (T) at time 0. Greaterthan or equal to 50% TGI over one tumor volume doubling time (TVDT) isconsidered an active anti-tumor response. TVDT is measured over thelinear growth range of the tumor, generally between 250 mm³-1000 mm³tumor size.Tumor Xenograft Study in A673 Model.

(1) Tumor Cell Line.

Human A673 Ewing Sarcoma cells (CRL-1598) were cultured following theinstructions recommended by ATCC. Briefly, cells were maintained in basemedium Dulbecco's Modified Eagle's Medium, (Invitrogen, Carlsbad,Calif.), supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad,Calif.) at 37° C. under a 5% CO₂ atmosphere.

(2) Xenograft Model.

10×10⁶ cells resuspended in 0.1 ml of PBS were injected subcutaneouslyin the dorsal flank region of female, seven to eight-week old, femaleathymic NCr nude mice (Taconic Farms, Hudson, N.Y.). Mice were housed inmicroisolator cages under HEPA filtered-air and temperature controlledbarrier racks. They were maintained on a 12 h light/dark cycle, fed withirradiated food and autoclaved water available ad libitum, and allowedto adapt to their new environment. Mice were manipulated using asepticprotocols and all experimental procedures and end point of tumor studiesfollowed institutional guidelines. Tumor implantation was monitoredusing caliper measurements to establish tumor growth rate beforerandomization and mice were randomized when tumors reached an average of150-200 mm³. Mice were euthanized when tumors reached a volume of 3,000mm³.

(3) Treatments.

Mice from each study were assigned to treatment groups described belowand received intraperitoneal administration at the regimens indicatedbelow. The vehicle group received a mono-specific ¹⁰Fn3-based VEGFR-2binder (Peg-V2Bshort, SEQ ID NO: 28) in reconstitution buffer: 10 mMSodium Acetate, 150 mM NaCl, pH 4.5. A mono-specific ¹⁰Fn3-based IGF-IRbinder (AT580-PEG40, SEQ ID NO: 27) was formulated in PBS and pegylated,his-tagged 385A08-Fn-V2B-cys (SEQ ID NO: 9) was formulated in 50 mMSodium Acetate, 150 mM NaCl, pH, 4.5. For the study described in FIG.26, there were six treatment groups, each consisting of 9 mice that weredosed on a three times a week (TIW) schedule of either vehicle, 50 mg/kg(mpk) of Peg-V2Bshort, 50 mpk of AT580-PEG40, 50 mpk of pegylated,his-tagged 385A08-Fn-V2B-cys, 100 mpk of pegylated, his-tagged385A08-Fn-V2B-cys, or a co-dose of 50 mpk of AT580-PEG40 and 50 mpk ofPeg-V2Bshort. In the dose response study discussed above (data notshown), there were five treatment groups, each consisting of 9 mice thatwere dosed on a TIW schedule of either vehicle, or 20, 60, 100, or 200mpk of pegylated, his-tagged 385A08-Fn-V2B-cys.

(4) Tumor Volume Measurements.

Tumor volume measurements were performed twice a week using Vernierscale calipers and tumor volumes (mm³) were calculated using theellipsoid formula [n/6 (L×W²)], where L represents the largest tumordiameter (mm) and W represents the smallest tumor diameter (mm). Tumormeasurements were noted as absolute values. Animal body weights wererecorded twice a week during the course of the experiment.

(5) Tumor Growth Inhibition (TGI).

TGI was calculated as the percent tumor growth of treated (T) groupsfrom control group (vehicle, C). Tumor growth was calculated bysubtracting initial tumor volume (at day 0) from the final tumor volumeat the end of the experiment. TGI=(1−[T−T₀]/[C−C₀])*100.

PK/PD Study Methods for Mouse and Monkey Models

Determination of Concentration in Serum or Plasma

A quantitative electrochemiluminescence assay was developed to detectthe concentration of 385A08-Fn-V2B-Cys (with and without his tag) inserum or plasma samples. In this assay, a mouse monoclonal antibodyspecific to the VEGFR2 portion of the molecule was adsorbed to astandard Meso Scale Discovery plate to capture the adnectin overnight at4° C. The serum/plasma sample was added to the plate for one hourincubation at room temperature. The captured adnectin was detected by arabbit polyclonal antibody specific to the scaffold region of theadnectin which was co-mixed with a goat anti-rabbit antibody linked witha SULFO-TAG, and added to the plate simultaneously. Following a wash toremove any unbound S ULFO-TAG reagent, a read buffer is added to eachwell and electrocheemiluminescense detection is used to detect bindingevents. 385A08-Fn-V2B-Cys (with and without his tag) plasma/serumconcentrations in samples were calculated based on the signal of thesample compared to a 4 parameter fit of a standard curve of385A08-Fn-V2B-Cys (with and without his tag).

Determination of VEGF-A Concentrations in Mice and Monkeys

A quantitative electrochemiluminescence assay for human and murineVEGF-A from Meso Scale Discovery (Gaithersburg, Md.) was employed todetect VEGF-A plasma concentration according to the manufacturer'sinstructions. Specific anti-VEGF-A monoclonal antibodies are pre-coatedonto Meso Scale Discovery (MSD) plates. The plate is blocked overnightat 4° C. and VEGF-A present in the standards, QCs and samples arecaptured during a 2 hour incubation step by the immobilized antibody.After washing away any unbound substances, a SULFO-TAG linked polyclonaldetection antibody is added to the wells for 2 hours. Following a washto remove any unbound SULFO-Tag reagent, a read buffer is added to eachwell and electrochemiluminescense detection is used to detect bindingevents. Plasma VEGF-A concentrations in samples were calculated based onthe signal of the sample compared to a 4 parameter fit of a standardcurve.

In Vivo Methods—Mouse

Single-Dose Pharmacokinetics in Mice.

The pharmacokinetics of his-tagged version of 385A08-Fn-V2B-Cys wasstudied in nude mice. Four groups of non-fasted animals (N=3-4 pergroup, 20-25 g) received his-tagged version of 385A08-Fn-V2B-Cys eitheras an intravenous (IV) bolus dose via the tail vein (5 mL/kg) or as anintraperitoneal (IP) dose (10 mL/kg). The doses were 5 and 50 mg/kg forboth routes. Prior to dosing, the compound was diluted from a stocksolution to an appropriate dosing solution concentration in phosphatebuffered saline (PBS). Serial blood samples (˜0.05 mL) were obtained bynicking the lateral tail vein. For the IV route, the sampling timepoints were 0.08, 0.5, 1, 2, 6, 12, 24, 48, 96, and 171 h post dose. Forthe IP route, the sampling time points were 0.5, 1, 2, 6, 12, 24, 48,96, 124, and 171 h post dose. Plasma samples were harvested by dilutingblood sample into a citrate phosphate dextrose solution in a 1:1 ratioand were stored at −20° C. until analysis.

Single-Dose Pharmacokinetic/Pharmacodynamic Study in Nude Mice Bearingthe Rh41 Tumor.

His-tagged version of 385A08-Fn-V2B-Cys was dosed intraperitoneally tonon-fasted nude mice (20-25 g) bearing the Rh41 tumor at doses of 20 and200 mg/kg along with a vehicle group. Prior to dosing, the drugcandidate was diluted from a stock solution to an appropriate dosingsolution concentration in phosphate buffered saline (PBS). Blood sampleswere obtained by cardiac puncture at 1, 3, 6, 16, 24, 32, 48, and 80 hpost dose in the drug-treated groups and 1, 24, and 36 post dose in thevehicle group. Serum samples were obtained after coagulation and storedat −20° C. until analysis for drug and VEGF-A concentrations.

Single-Dose Pharmacokinetic/Pharmacodynamic Study in Nude Mice Bearingthe A673 Tumor.

385A08-Fn-V2B-Cys (non-his tagged) was dosed intraperitoneally tonon-fasted nude mice (20-25 g) bearing the A673 tumor at doses of 20 and200 mg/kg. Prior to dosing, the drug candidate was diluted from a stocksolution to an appropriate dosing solution concentration in PBS. Plasmasamples were harvested by cardiac puncture into sodium heparin coated BDvacutainer tubes at 1, 3, 6, 16, 24, 32, 48, 72, 96, and 120 h postdose, and were stored at −20° C. until analysis for drug and VEGF-Aconcentrations.

In Vivo Methods—Monkey

Single-Dose Pharmacokinetic/Pharmacodynamic Study in Monkeys.

The pharmacokinetics of his-tagged version of 385A08-Fn-V2B-Cys wasevaluated in male cynomolgus monkeys. Following an overnight fast, 2animals (˜4 kg) received his-tagged version of 385A08-Fn-V2B-Cys atdoses of 3 and 30 mg/kg by a constant-rate IV infusion (5 mL/kg) for 10min via the femoral vein. Prior to dosing, the drug candidate wasdiluted from a stock solution to an appropriate dosing solutionconcentration in PBS. Blood samples (˜1 mL) were collected into K₂EDTAtubes from the femoral artery at predose and 0.17, 0.25, 0.5, 1, 2, 4,6, 8, 24, 30, 54, 72 (day 3), 96 (day 4), 168 (day 7), 216 (day 9), 264(day 11), 336 (day 14), 384 (day 16), 432 (day 18), 504 h (day 21) postdose. Plasma samples were obtained after centrifugation at 4° C.(1500-2000×g) and stored at ˜20° C. until analysis for drug and VEGF-Aconcentrations.

Using the same study design, 385A08-Fn-V2B-Cys (non-his tagged) wasstudied at 3 mg/kg following IV administration to male cynomolgusmonkeys (N=3, ˜4 kg). After the 3-week study and additional 3-weekwashout period, 385A08-Fn-V2B-Cys (non-his tagged) was re-dosed to thesame monkeys at the same dose, with blood samples collected up to a weekpost dose.

Data Analysis

The data are expressed as mean±standard deviation (SD).

The pharmacokinetic parameters of 385A08-Fn-V2B-Cys (his tagged andnon-his tagged) were obtained by non-compartmental analysis of plasma(serum) concentration vs. time data (KINETICA™ software, Version 4.2,InnaPhase Corporation, Philadelphia, Pa.). The peak concentration (Cmax)and time for Cmax were recorded directly from experimental observations.The area under the curve from time zero to the last sampling time(AUC(0-T)) and the area under the curve from time zero to infinity(AUC(INF)) were calculated using a combination of linear and logtrapezoidal summations. The total plasma clearance (CLTp), steady-statevolume of distribution (Vss), terminal half-life (T1/2) and meanresidence time (MRT) were estimated after IV administration. Estimationsof AUC and T1/2 were made using a minimum of 3 time points withquantifiable concentrations.

The pharmacokinetic (PK) and pharmacodynamic (PD) data of385A08-Fn-V2B-Cys (his tagged and non-his tagged) generated in mice andmonkeys were modeled using the SAAM II (version 1.2.1, Seattle, Wash.).For the mouse PK data obtained after IV and IP doses of 5 and 50 mg/kg,the naive-pooled serum concentration-time data were simultaneouslyfitted using a two-compartment model coupled with first-order absorptionkinetics.

The PD response, measured as either mVEGF-A or hVEGF-A concentrations inplasma or serum, was modeled using an indirect response model, with theassumptions that the production of VEGF-A was not altered in thepresence of 385A08-Fn-V2B-Cys (his tagged and non-his tagged) andbinding of VEGF-A to VEGFR-2, as its major clearance pathway, wasblocked by 385A08-Fn-V2B-Cys (his tagged and non-his tagged).

We claim:
 1. A method of treating cancer comprising administering to asubject in need thereof a therapeutically effective amount of apolypeptide comprising: (A)(a) an N-terminal domain comprising a firstfibronectin type III tenth domain (¹⁰Fn3), wherein the ¹⁰Fn3 domain (i)comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop, EF;and a loop, FG; (ii) has at least one loop selected from loop BC, DE,and FG with an altered amino acid sequence relative to the sequence ofthe corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO: 1); and(iii) binds to insulin-like growth factor-I receptor (IGF-IR) with aK_(D) of less than 500 nM; and (b) a C-terminal domain comprising asecond fibronectin type III tenth domain (¹⁰Fn3), wherein the ¹⁰Fn3domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; aloop, EF; and a loop, FG; (ii) has at least one loop selected from loopBC, DE, and FG with an altered amino acid sequence relative to thesequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO:1); and (iii) binds to vascular endothelial growth factor receptor 2(VEGFR2) with a K_(D) of less than 500 nM; wherein the first and second¹⁰Fn3 domains are linked via a polypeptide selected from aglycine-serine based linker, a glycine-proline based linker, aproline-alanine based linker or the amino acid sequence of SEQ ID NO:20, wherein the polypeptide comprises an amino acid sequence at least85% identical to the amino acid sequence of any one of SEQ ID NOs: 8-11,29-31, and 63-70; (B)(a) an N-terminal domain comprising a first ¹⁰Fn3domain, wherein the ¹⁰Fn3 domain (i) comprises a loop, AB; a loop, BC; aloop, CD; a loop, DE; a loop, EF; and a loop, FG; (ii) has at least oneloop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain (SEQ ID NO: 1); (iii) binds to VEGFR2 with a K_(D) of lessthan 500 nM; and (iv) is at least 85% identical to SEQ ID NO: 40; and(b) a C-terminal domain comprising a second ¹⁰Fn3 domain, wherein the¹⁰Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop,DE; a loop, EF; and a loop, FG; (ii) has at least one loop selected fromloop BC, DE, and FG with an altered amino acid sequence relative to thesequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO:1); (iii) binds to IGF-IR with a K_(D) of less than 500 nM; and (iv) isat least 85% identical to SEQ ID NO: 35; wherein the first and second¹⁰Fn3 domains are linked via a polypeptide selected from aglycine-serine based linker, a glycine-proline based linker, aproline-alanine based linker or the amino acid sequence of SEQ ID NO:20; (C)(a) an N-terminal domain comprising a first ¹⁰Fn3 domain, whereinthe ¹⁰Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; aloop, DE; a loop, EF; and a loop, FG; (ii) has at least one loopselected from loop BC, DE, and FG with an altered amino acid sequencerelative to the sequence of the corresponding loop of the human ¹⁰Fn3domain (SEQ ID NO: 1); (iii) binds to IGF-IR with a K_(D) of less than500 nM; and (iv) is at least 85% identical to SEQ ID NO: 35; and (b) aC-terminal domain comprising a second ¹⁰Fn3 domain, wherein the ¹⁰Fn3domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; aloop, EF; and a loop, FG; (ii) has at least one loop selected from loopBC, DE, and FG with an altered amino acid sequence relative to thesequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO:1); (iii) binds to VEGFR2 with a K_(D) of less than 500 nM; and (iv) isat least 85% identical to SEQ ID NO: 40; wherein the first and second¹⁰Fn3 domains are linked via a polypeptide selected from aglycine-serine based linker, a glycine-proline based linker, aproline-alanine based linker or the amino acid sequence of SEQ ID NO:20; (D)(a) an N-terminal domain comprising a first ¹⁰Fn3 domain whichbinds to IGF-IR, wherein the ¹⁰Fn3 domain comprises BC, DE, and FG loopscomprising the amino acid sequence set forth in SEQ ID NOs: 2, 3, and 4,respectively; and (b) a C-terminal domain comprising a second ¹⁰Fn3domain which binds to VEGFR2, wherein the second ¹⁰Fn3 domain comprisesBC, DE, and FG loops comprising the amino acid sequence set forth in SEQID NOs: 5, 6, and 7, respectively; or (E)(a) an N-terminal domaincomprising a first ¹⁰Fn3 domain which binds to VEGFR2, wherein the ¹⁰Fn3domain comprises BC, DE, and FG loops comprising the amino acid sequenceset forth in SEQ ID NOs: 5, 6, and 7, respectively; and (b) a C-terminaldomain comprising a second ¹⁰Fn3 domain which binds to IGF-IR, whereinthe second ¹⁰Fn3 domain comprises BC, DE, and FG loops comprising theamino acid sequence set forth in SEQ ID NOs: 2, 3, and 4, respectively.2. The method of claim 1, wherein the first and/or second ¹⁰Fn3 domaincomprises an N-terminal extension sequence selected from the groupconsisting of M, MG, G, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 48.3. The method of claim 1, wherein the first and/or second ¹⁰Fn3 domainis linked at its C-terminus to the amino acid sequence of E, EI, ES, EC,EGS, EGC, EID, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 71, or SEQ ID NO:
 72. 4.The method of claim 1, wherein the polypeptide further comprises one ormore pharmacokinetic (PK) moieties selected from the group consistingof: a polyoxyalkylene moiety, a human serum albumin binding protein,sialic acid, human serum albumin, IgG, an IgG binding protein,transferrin, and an Fc fragment.
 5. The method of claim 4, wherein thePK moiety and the polypeptide are linked via at least one disulfidebond, a peptide bond, a polypeptide, a polymeric sugar, or apolyethylene glycol moiety.
 6. The method of claim 1, wherein thepolypeptide has been deimmunized to remove one or more T-cell epitopes.7. The method of claim 1, wherein the IGF-IR binding ¹⁰Fn3 domain bindsto IGF-IR with a KD of less than 500 nM.
 8. The method of claim 1,wherein the VEGFR2 binding ¹⁰Fn3 domain binds to VEGFR2 with a KD ofless than 500 nM.
 9. The method of claim 1, wherein the polypeptidecomprises: (a) an N-terminal domain comprising a first ¹⁰Fn3 domain,wherein the ¹⁰Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop,CD; a loop, DE; a loop, EF; and a loop, FG; (ii) has at least one loopselected from loop BC, DE, and FG with an altered amino acid sequencerelative to the sequence of the corresponding loop of the human ¹⁰Fn3domain (SEQ ID NO: 1); and (iii) binds to IGF-IR with a K_(D) of lessthan 500 nM; and (b) a C-terminal domain comprising a second ¹⁰Fn3domain, wherein the ¹⁰Fn3 domain (i) comprises a loop, AB; a loop, BC; aloop, CD; a loop, DE; a loop, EF; and a loop, FG; (ii) has at least oneloop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain (SEQ ID NO: 1); and (iii) binds to VEGFR2 with a K_(D) ofless than 500 nM; wherein the first and second ¹⁰Fn3 domains are linkedvia a polypeptide selected from a glycine-serine based linker, aglycine-proline based linker, a proline-alanine based linker or theamino acid sequence of SEQ ID NO: 20, wherein the polypeptide comprisesan amino acid sequence at least 85% identical to the amino acid sequenceof any one of SEQ ID NOs: 8-11, 29-31, and 63-70.
 10. The method ofclaim 1, wherein the polypeptide comprises: (a) an N-terminal domaincomprising a first ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain (i) comprisesa loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop, EF; and a loop,FG; (ii) has atleast one loop selected from loop BC, DE, and FG with analtered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO: 1); (iii) bindsto VEGFR2 with a K_(D) of less than 500 nM; and (iv) is at least 85%identical to SEQ ID NO: 40; and (b) a C-terminal domain comprising asecond ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain (i) comprises a loop, AB;a loop, BC; a loop, CD; a loop, DE; a loop, EF; and a loop, FG; (ii) hasat least one loop selected from loop BC, DE, and FG with an alteredamino acid sequence relative to the sequence of the corresponding loopof the human ¹⁰Fn3 domain (SEQ ID NO: 1); (iii) binds to IGF-IR with aK_(D) of less than 500 nM; and (iv) is at least 85% identical to SEQ IDNO: 35; wherein the first and second ¹⁰Fn3 domains are linked via apolypeptide selected from a glycine-serine based linker, aglycine-proline based linker, a proline-alanine based linker or theamino acid sequence of SEQ ID NO:
 20. 11. The method of claim 1, whereinthe polypeptide comprises: (a) an N-terminal domain comprising a first¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain (i) comprises a loop, AB; a loop,BC; a loop, CD; a loop, DE; a loop, EF; and a loop, FG; (ii) has atleast one loop selected from loop BC, DE, and FG with an altered aminoacid sequence relative to the sequence of the corresponding loop of thehuman ¹⁰Fn3 domain (SEQ ID NO: 1); (iii) binds to IGF-IR with a K_(D) ofless than 500 nM; and (iv) is at least 85% identical to SEQ ID NO: 35;and (b) a C-terminal domain comprising a second ¹⁰Fn3 domain, whereinthe ¹⁰Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; aloop, DE; a loop, EF; and a loop, FG; (ii) has at least one loopselected from loop BC, DE, and FG with an altered amino acid sequencerelative to the sequence of the corresponding loop of the human ¹⁰Fn3domain (SEQ ID NO: 1); (iii) binds to VEGFR2 with a K_(D) of less than500 nM; and (iv) is at least 85% identical to SEQ ID NO: 40; wherein thefirst and second ¹⁰Fn3 domains are linked via a polypeptide selectedfrom a glycine-serine based linker, a glycine-proline based linker, aproline-alanine based linker or the amino acid sequence of SEQ ID NO:20.
 12. The method of claim 1, wherein the polypeptide comprises: (a) anN-terminal domain comprising a first ¹⁰Fn3 domain which binds to IGF-IR,wherein the ¹⁰Fn3 domain comprises BC, DE, and FG loops comprising theamino acid sequence set forth in SEQ ID NOs: 2, 3, and 4, respectively;and (b) a C-terminal domain comprising a second ¹⁰Fn3 domain which bindsto VEGFR2, wherein the second ¹⁰Fn3 domain comprises BC, DE, and FGloops comprising the amino acid sequence set forth in SEQ ID NOs: 5, 6,and 7, respectively.
 13. The method of claim 12, wherein the first ¹⁰Fn3domain and second ¹⁰Fn3 domain are linked via a polypeptide selectedfrom a glycine-serine based linker, a glycine-proline based linker, aproline-alanine based linker, or a polypeptide comprising the amino acidsequence of SEQ ID NO:
 20. 14. The method of claim 1, wherein thepolypeptide comprises: (a) an N-terminal domain comprising a first ¹⁰Fn3domain which binds to VEGFR2, wherein the ¹⁰Fn3 domain comprises BC, DE,and FG loops comprising the amino acid sequence set forth in SEQ ID NOs:5, 6, and 7, respectively; and (b) a C-terminal domain comprising asecond ¹⁰Fn3 domain which binds to IGF-IR, wherein the second ¹⁰Fn3domain comprises BC, DE, and FG loops comprising the amino acid sequenceset forth in SEQ ID NOs: 2, 3, and 4, respectively.
 15. The method ofclaim 14, wherein the first ¹⁰Fn3 domain and second ¹⁰Fn3 domain arelinked via a polypeptide selected from a glycine-serine based linker, aglycine-proline based linker, a proline-alanine based linker, or apolypeptide comprising the amino acid sequence of SEQ ID NO: 20.